Octamer Formation and Stability in a Mitochondrial Creatine Kinase from a Protostome Invertebrate Gregg G

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2005 Octamer Formation and Stability in a Mitochondrial Creatine Kinase from a Protostome Invertebrate Gregg G. Hoffman

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COLLEGE OF ARTS AND SCIENCES

OCTAMER FORMATION AND STABILITY IN A MITOCHONDRIAL CREATINE KINASE FROM A PROTOSTOME INVERTEBRATE

GREGG G. HOFFMAN

A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Degree Awarded: Fall Semester, 2005

The members of the Committee approve the Dissertation of Gregg G. Hoffman defended on October 24, 2005.

W. Ross Ellington Professor Directing Dissertation

Michael S. Chapman Outside Committee Member

P. Bryant Chase Committee Member

Timothy S. Moerland Committee Member

Kenneth H. Roux Committee Member

Approved:

Timothy S. Moerland, Chair, Department of Biological Science

The Office of Graduate Studies has verified and approved the above named committee members.

ii TABLE OF CONTENTS

List of Tables ...... Page iv List of Figures ...... Page v List of Abbreviations ……………………………………………………………Page vii Abstract ……………………………………………………………..Page ix

INTRODUCTION ...... Page 1

MATERIALS and METHODS...... Page 12

RESULTS ………………………………………………………………Page 22

DISCUSSION ………………………………………………………………Page 62

LITERATURE CITED………………………………………………………….Page 71

BIOGRAPHICAL SKETCH……………………………………………………Page 80

iii LIST OF TABLES

Table 1: Mutagenesis primer sequences ...... Page 20

Table 2: Impact of temperature on octamer / dimer equilibrium...... Page 34

Table 3: Percent identity and similarity...... Page 44

Table 4: Ramachandran plot statistics for the homology model...... Page 48

Table 5: Comparitive validation statistics for the homology model...... Page 51

Table 6: Residues at the dimer / dimer interface...... Page 55

Table 7: Hydrodynamic radius, %PD, and specific activity ...... Page 61

iv LIST OF FIGURES

Figure 1: The structures of the eight phosphagens ...... Page 3

Figure 2: The role of MtCK within mitochondria ...... Page 9

Figure 3: Cloning and expression flow chart...... Page 14

Figure 4: SDS-PAGE of raw cell lysates...... Page 23

Figure 5: UV and enzyme activity profiles – protein purification...... Page 24

Figure 6: SDS-PAGE of CVMtCK purification steps ...... Page 25

Figure 7: Representative UV profile - Superdex 200HR FPLC ...... Page 26

Figure 8: Demonstration of octamer / dimer interconvertibility...... Page 28

Figure 9: Effect of protein concentration on oligomeric state ...... Page 29

Figure 10: Effect of temperature on oligomeric state and kdis ...... Page 30

Figure 11: Time course of octamer dissociation at two temperatures ...... Page 32

Figure 12: Thermal jump study...... Page 33

Figure 13: Effect of TSAC - conversion on octamer stability...... Page 36

Figure 14: DLS standard curve...... Page 38

Figure 15: DLS % octamer curve ...... Page 39

Figure 16: Effect of concentration on octamer / dimer equilibrium ...... Page 40

Figure 17: Representative DLS profile...... Page 41

Figure 18: Multiple sequence alignment...... Page 43

Figure 19: CVMtCK homology model...... Page 45

Figure 20: Ramachandran plots ...... Page 47

Figure 21: RMS deviation between CVMtCK model and template...... Page 49

Figure 22: Surface representations showing dimer / dimer interface ...... Page 54

Figure 23: Multiple sequence alignment of residues around Trp264 ...... Page 58

Figure 24: Relative orientation of Trp264 ...... Page 59

vi LIST OF ABBREVIATIONS

ADP adenosine diphosphate

ATP adenosine triphosphate

ANT adenine nucleotide translocase

AK arginine kinase

BBCK brain type cytoplasmic creatine kinase

-MSH beta - mercaptoethanol

CK creatine kinase (CK)

CM Carboxymethyl

Cr creatine (Cr)

CVMtCK Chaetopterus variopedatus mitochondrial creatine kinase

DLS dynamic light scattering

DTT dithiothreitol

∆GATP effective free energy of ATP hydrolysis

GK glycocyamine kinase

HTK hypotaurocyamine kinase

IMS intermembrane space

IPTG isopropyl--D-thiogalactopyranoside

LB Luria broth

LK lombricine kinase

MMCK muscle type cytoplasmic creatine kinase

Mr relative molecular mass

vii MtCK mitochondrial CK

OK opheline kinase

PCr phosphocreatine

%PD polydispersity

Pi inorganic phosphate

PK phosphagen kinase

PTP permeability transition pore

SarMtCK sarcomeric mitochondrial creatine kinase

ThK thalassemine kinase

TK taurocyamine kinase

TSAC transition state analog complex

Rh hydrodynamic radius

SPDBV SwissPDB Viewer

UbiMtCK ubiquitous mitochondrial creatine kinase

viii ABSTRACT

The free energy yield from ATP hydrolysis is directly related to the [ATP]/

([ADP] [Pi]) ratio within any given cell, and it has long been clear that cells can not maintain functionality when this ratio undergoes a significant change. This energetic challenge is most pronounced within the cells of excitable tissues with high and variable energy demands, cells such as cardiomyocytes, neurons, transport epithelia, and primitive free swimming spermatozoa. It is within these very cells that the creatine kinase (CK) system comes into play. CK catalyzes the reversible transfer of the -phosphoryl group

from MgATP to the guanidine group of creatine, thereby maintaining ∆GATP by buffering

the [ATP]/([ADP] [Pi]) ratio when there are temporal and spatial mismatches of ATP supply and ATP demand. Three distinct CK gene families exist – mitochondrial, cytoplasmic and flagellar – each targeted to different intracellular compartments. These genes appear to have evolved at the dawn of the radiation of multicellular animals. As part of on-going efforts to probe the evolutionary physiology in the CK gene family, the cDNAs for mitochondrial CKs from the protostome polychaete Chaetopterus variopedatus (CVMtCK) and chicken cardiac tissue (SarMtCK) have been cloned, inserted into an expression vector and recombinant protein expressed, purified and characterized. Recombinant CVMtCK was primarily octameric as was the well- characterized chicken SarMtCK. Using two independent methods (size-exclusion chromatography and dynamic light scattering, or DLS), studies of oligomerization dynamics showed that CVMtCK exhibited the same reversible transition between octamers and dimers as has been reported for MtCKs from higher organisms, and that these ancient octamers displayed the same dissociation and reassociation profile as that seen in the MtCKs from birds and man under various thermal and concentration regimes. However, the rate of change in both directions is much more rapid for CVMtCK. Interestingly, and perhaps importantly, when CVMtCK was converted to the transition - state analog complex (TSAC) in the presence of NO3 , MgADP, and creatine, both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. To

ix evaluate the potential structural correlates of the observed differences in octamer stability, a homology model was developed using the octameric crystal structures of SarMtCK and human ubiquitous UbiMtCK as templates. The resulting model was validated by a variety of on-line tools. Comparison of the structures showed some differences in the interactions occurring across the dimer - dimer interface which are likely to impact the stability of the octameric structure. In all structures, a key and absolutely conserved tryptophan residue is present in this interface. Site-directed mutagenesis procedures were employed to mutate this Trp residue to Cys, Phe, Leu and Tyr. In all cases, the specific activity was unaffected but the recombinant protein was dimeric; no octameric protein was detected using chromatography or DLS. The overall results of this effort show that octamer formation is a primitive character of MtCKs and that there has been some fine-tuning – in an evolutionary sense – of the nature of the interactions promoting and stabilizing the octameric state.

x INTRODUCTION

In typical healthy cells the concentration of adenosine triphosphate (ATP) is nearly universally homeostatic; that is, changes in the rate of ATP utilization do not commonly result in large changes in ATP concentrations. Liver, and many other tissues, routinely experience a 1.5 to 2.0-fold difference between the resting ATP turnover rate and the rate seen in the ‘activated’ state (Hochachka & McClelland, 1997). On the other hand, vertebrate cardiac myocytes are reported to consume 2 % of the available ATP pool during a single heartbeat (Cortassa et al., 2003), and many skeletal muscle myocytes must operate efficiently during 100-fold changes in ATP turnover rates (Hochachka & McClelland, 1997). While many metabolites are maintained within narrow concentration ranges, none, with the possible exception of oxygen, appears to be as precisely controlled as ATP. These observations serve to highlight a central question in the study of cellular metabolism - given that cellular “work” would almost certainly require drastic changes in intracellular conditions, and these changes must somehow be linked to the intensity of the work being performed, how then can cells with highly variable work loads achieve (and maintain) a precise metabolic homeostasis in the concentration of ATP and at the same time exercise precise control over this same metabolism? The physiological impact of this ATP turnover is not strictly a function of the absolute ATP concentrations available within a given cell but rather it is a function of the ATP chemical potential, also known as the effective free energy of ATP hydrolysis, or

∆GATP. The ∆GATP is dependent on the concentration of ATP, adenosine diphosphate

(ADP) and inorganic phosphate (Pi) as described by Gibbs in the following equation (R = molar gas constant: 8.314 m2·kg·s-1·K-1·mol-1, T = temperature in K):

∆GATP = ∆G°’ATP + R T ln ([ADP] [Pi] / [ATP])

The free energy yield from ATP hydrolysis under standard conditions (∆G°’ATP) is approximately -30.5 kJ/mol, far less than the energy required to maintain even baseline levels of the electro-chemical potentials required for cellular function. Typically,

1 oxidative phosphorylation and glycolysis displace this reaction far from equilibrium and

∆GATP is maintained between -55 and -64 kJ/mol in excitable tissues with high and variable energy demands such as muscle cells and neurons and between -45 and -51 kJ/mol in other non-excitable tissues (Kammermeier, 1993). As is indicated above, maintenance of a highly negative ∆GATP can quickly become problematic when ATP is utilized and ADP and Pi production rises.

It has long been clear that cells cannot maintain functionality when ∆GATP undergoes a significant change (Seraydarian, 1975), and it is in excitable cells that are facing energetic challenges that this problem becomes most pronounced. It is here, within the cells of these excitable tissues facing high and/or variable energy demands, that the phosphagen – phosphagen kinase (PK) systems come into play. Phosphagens are phosphorylated guanidine-containing compounds that act to buffer the value of ∆GATP in cells that exhibit a need for highly efficient storage of the energy derived from substrate oxidation (Kammermeier, 1987). This is most pronounced when such cell-types are experiencing a disequilibrium between ATP hydrolysis (demand) and ATP synthesis (supply) (Meyer et al., 1984). PKs act according to the reaction below:

Phosphagen + MgADP + H+ guanidine acceptor + MgATP

As can be seen, this phosphagen kinase reaction will serve to maintain highly negative values of ∆GATP during periods of metabolic activity when MgATP utilization exceeds the locally available MgATP supply by “on-site” re-phosphorylation of ADP as it is produced, consuming protons in the process. Eight different and highly conserved (PKs all share at least 40% identity) phosphagen kinase systems have been reported from within the cells of organisms across virtually all metazoan phyla, each with a unique corresponding phosphagen (Fig. 1). These are creatine kinase (CK), arginine kinase (AK), glycocyamine kinase (GK), taurocyamine kinase (TK), hypotaurocyamine kinase (HTK), lombricine kinase (LK), opheline kinase (OK), and thallassemine kinase (ThK). Of these, the creatine kinase and arginine kinase systems are the most widespread and studied, and CK is the only phosphagen kinase system found in, but it is not limited to, the craniates (hagfish and true

NH-PO3NH2 NH-PO3NH2 HN=C HN=C

NH-(CH2)3-CH-CO2H N-CH2-CH-CO2H

NH2 CH3

Arginine Phosphate (AP) Creatine Phosphate (CP)

NH-PO3NH2 NH-PO3NH2 HN=C HN=C

NH-CH2-CO2H NH-(CH2)2-SO2H

Glycocyamine Phosphate (GP) Hypotaurocyamine Phosphate (HTP)

NH-PO3NH2 NH-PO3NH2 HN=C O HN=C O

NH-(CH2)2-O-P-O-CH-CO2H NH-(CH2)2-O-P-O-CH3

OH NH2 OH

Lombricine Phosphate (LP) Opheline Phosphate (OP)

NH-PO3NH2 NH-PO3NH2 HN=C HN=C O

NH-(CH2)2-SO3H NH-(CH2)2-O-P-O-CH2-CH-CO2H

OH N-(CH3)2

Taurocyamine Phosphate (TP) Thalassemine Phosphate (ThP)

Figure 1. The structures of the eight phosphagens (from Ellington, 2001).

3 vertebrates) [for a review of phosphagen kinase distribution, see (Ellington, 2001)]. It is this creatine kinase system that is the primary focus of the work presented here. Creatine kinase catalyzes the reversible transfer of the -phosphoryl group from MgATP to the guanidine group of creatine (Cr) forming the phosphagen phosphocreatine (PCr) in a random order bi-bi reaction according to the scheme listed below:

MgADP + PCr + H+ MgATP + Cr

Specifically, CK plays a central role in energy homeostasis within cells that undergo high and highly variable rates of ATP turnover such as neurons, muscle fibers, transport epithelia, and spermatozoa, and is widely distributed in both invertebrate and vertebrate groups (Ellington, 2001). Historically, the CK system has been implicated in a number of “energy- management” roles within cells, specifically in countering miss-matches between ATP demand and ATP supply. The most widely accepted of these is that of a “temporal ATP

buffer” whereby CK maintains a highly negative ∆GATP by buffering the [ATP] / ([ADP]

[Pi]) ratio during periods of temporal disequilibrium between ATP production and utilization (Meyer et al., 1984; Kammermeier, 1987, 1993), such as might take place in burst muscle contraction. A second role for the CK system, which Bessman and Geiger (1981) called a “phosphocreatine shuttle”, holds that the PCr and Cr serve to metabolically connect sites of energy production, or “sources”, with sites of energy utilization, or “sinks”, via separate CK isoforms localized, or compartmentalized, at a sub-cellular level (Gercken & Schlette, 1968; Jacobus et al., 1983; Bessman & Carpenter, 1985; Jacobus, 1985; Wallimann et al., 1985; Ventura-Clapier et al., 1987). Meyer et al. (1984) re-evaluated the so-called “shuttle” and reasoned that this role is merely a special application of the temporal ATP buffering role of CK. They referred to this as spatial ATP buffering and concluded that it is the result of the following features of the CK system: (a) PCr (and Cr) concentrations are in excess relative to adenine nucleotides concentrations within the cell, (b) the CK reaction has a high K’ ([ATP] [Cr] / [ADP]

[PCr]), (c) the diffusion coefficient for PCr (DPCr) is greater than DATP, and (d) the CK

4 activity is sufficiently high to maintain the reaction at near equilibrium with its substrates in vivo. A later incarnation of the shuttle (Bessman & Geiger, 1981), and the more recent phosphocreatine circuit (Wallimann et al., 1992) models, have even more controversial features. Here CK is postulated to maintain local [ATP] / ([ADP] [Pi] ) ratios on a subcellular level as a result of its “functional coupling” to ATP-consuming and ATP- producing sites. It has been pointed out that CK can serve to enhance the thermodynamic efficiency of both ATP hydrolysis and synthesis via its ability to regulate the ratio of ATP: ADP at the sources and at the sinks within the cell (Jacobus & Lehninger, 1973; Saks et al., 1976; Matthews et al., 1982; Wallimann & Eppenberger, 1985; Wallimann et al., 1989; Wallimann & Eppenberger, 1990). This controversy can be summarized in the following way: cells can either (a) maintain globally high CK levels throughout the cytoplasm (Meyer et al., 1984) or, (b) target the CK enzyme system to specific local regions of ATP synthesis and to regions of hydrolysis (Wallimann et al., 1992), or (c), do both. In fact, as Meyer et al. (1984) pointed out, compartment-specific targeting of CK is not necessary for spatial ATP buffering but would serve to achieve an “economy of protein” by creating specific regions of high CK activity only where it is needed, thereby reducing the total protein required for CK to act as a spatial buffer for ATP throughout the cell. Recently, many new CK sequences have been reported, sometimes on a seemingly weekly basis, and analysis of these sequences indicates that there are three distinct families of CK genes, each specifically targeted to distinct compartments within cells. Suzuki and coworkers (2004) have shown that there is a family of CK genes targeted to the intermembrane space (IMS) of the mitochondria (MtCK), another targeted to the cytoplasm, and yet another, consisting of three fused and complete CK domains, which is targeted to the flagellar membranes of primitive-type spermatozoa. These three CK genes were the result of early gene duplication (followed, in the case of the flagellar genes, by gene fusion) and divergence events that occurred prior to the divergence of the protostomes and deuterostome lineages (Suzuki et al., 2004). Two additional gene duplication events took place around the radiation event that led to the chordates (Graber & Ellington, 2001; Uda et al., 2004), and subsequent divergence of these genes has resulted in four very distinct nuclear CK genes. These genes are expressed in a tissue-

5 specific manner, assume different quaternary structure, and are targeted to different and very specific intracellular compartments (Suzuki et al., 2004). In vertebrates, nuclear genes code for two cytoplasmic and two mitochondrial isoforms (Wallimann et al., 1992). One cytoplasmic isoform is specifically expressed in skeletal muscle (M type) and another in neuronal tissues, cardiac and smooth muscle, and other tissues (B type). These cytoplasmic isoforms exist as homo-dimers (BBCK and MMCK respectively) and can exist as heterodimers (MBCK) when expressed in the same cells (Wallimann et al., 1992). The mitochondrial CKs go one step further; they are expressed in a tissue-specific manner and they are directly and exclusively targeted to the mitochondrial IMS as a pre- protein via a cleavable N-terminal targeting sequence. Once within the IMS, MtCKs have been shown to have a high affinity for the cardiolipin rafts associated with the adenine nucleotide translocase (ANT) of the inner membrane (Stachowiak et al., 1996) and with porin in the outer membrane (Brdiczka, 1994). This binding capacity has important implications which will be discussed later in this narrative. Although MM- and BBCK are soluble and generally targeted to the cytoplasm, a significant fraction of both M and B-type CK is directly localized at sites of ATP hydrolysis. For example, MMCK is bound to chicken pectoralis myofibrils in sufficient quantities to phosphorylate the MgADP produced by the actin-activated ATPases (Wallimann et al., 1984) and this same group reported a significant body of evidence that indicated the MMCK is not only associated with the M band but is in fact an integral component of the M line structure in these myofibrils (Wallimann & Eppenberger, 1985). It has also been shown that BBCK is found in close association with acetylcholine receptors on the innervated ventral face of electrocytes from the electric ray Torpedo marmorata, where it is intimately associated with Na+/K+ ATPase and with ATP- dependent transmitter release at the nerve ending (Wallimann et al., 1985). In addition, M-type CK has been shown to be specifically bound to the sarcoplasmic reticulum in the vicinity of the Ca2+- ATPases where it was shown to exist in sufficient quantities to support, in the presence of PCr and MgADP, a significant fraction of the maximal in vitro Ca2+ uptake. This MgATP regeneration rate was demonstrated to be similar to the rate of Ca2+ - stimulated ATPase activity, indicating that CK activity is capable of supporting the

6 local ATP:ADP ratio in the absence of mitochondria (Rossi et al., 1990). Mitochondrial CK genes contain the code for an N-terminal transit peptide that targets the isoform to the IMS (Haas & Strauss, 1990) where it exists primarily as a homo-octamer. These octamers have been shown to exist in an equilibrium with their constituent dimers, at least in vitro (Lipskaia et al., 1985; Schlegel et al., 1988). One, sarcomeric (SarMtCK), is targeted to muscle cell mitochondria and the other, ubiquitous (UbiMtCK), is targeted to the mitochondrial IMS in neuronal and other tissues (Wallimann et al., 1992). It was the existence of these tissue-specific and, more importantly, compartment- specific isoforms of CK that has led the Wallimann group to strongly champion the model of CK–directed ∆GATP buffering known as the phosphocreatine shuttle first proposed by Gerchen in 1968 (Gercken & Schlette, 1968; Bessman & Geiger, 1981; Bessman & Carpenter, 1985; Bessman, 1987). It has also been suggested that MtCK molecules, because of their ability to bind to the cardiolipin rafts associated with ANT, may have preferential access to ATP as it exits the mitochondrial matrix (“the source”) where they catalyze the transfer of the phosphoryl group from MgATP to Cr (Muller et al., 1986; Dolder et al., 2001; Schlattner et al., 2004). The resulting PCr, because of its higher diffusion coefficient relative to ATP, would then diffuse into the cytoplasm where it is available to phosphorylate MgADP, re-forming MgATP at the sites of ATP hydrolysis (the “sink”) via the bound MM-and BB- type CKs, examples of which are discussed above. It was this localization, in combination with the relatively high diffusion coefficients of PCr and creatine relative to adenylate nucleotides, which led the Bessman group to propose this “phosphocreatine shuttle”. Here, adenine nucleotides can be thought to exist in two functionally separate pools, one pool within the mitochondria and functionally coupled to the sites of ATP production and the other in the cytoplasm where it is associated with the sites of ATP hydrolysis (Bessman & Geiger, 1981; Bessman & Carpenter, 1985; Bessman, 1987). Not only does the distinct and tissue-specific subcellular localization of different CK isoforms at the sites of energy production and energy utilization indicate that CK has an important functional role in cellular energy management, but even a cursory investigation of the CK literature will lead one to conclude that oligomerization also has

7 important implications, both functional and structural. While AK exists within cells as a monomer, cytoplasmic CKs exist as dimers and the mitochondrial isoforms of CK, in all but the most primitive of animals (Sona et al., 2004), form octamers. To date, there have been no reports of a naturally occurring monomeric CK. In fact, CK monomers exist in vitro only under non-physiological conditions (e.g. high concentrations of KI, urea, or guanidine hydrochloride) (Wyss et al., 1990; Gross et al., 1994). It is the MtCK octamers themselves that provided the strongest support for the “functional coupling” between adenine nucleotide pools and CK isoenzymes that was first reported by the Saks group as early as 1976 (Saks et al., 1976). An overview of this idea can be seen in Figure 2. This cartoon shows how MtCK octamers could mediate the formation of the contact sites mentioned above, and schematically indicates how the close association between octameric MtCK and the mitochondrial membranes in the vicinity of ANT and porin could provide MtCK with a sort of “preferential access” to newly formed molecules of ATP as they exit the matrix (Nicolay et al., 1990; Rojo et al., 1991; Schnyder et al., 1994). Octamers of MtCK are known to bind strongly to the phospholipids on the inner surface of the outer mitochondrial membrane and to cardiolipin on the outer surface of the inner membrane where they potentially mediate the formation of contact sites (regions of close apposition between the inner and outer mitochondrial membrane) (Schlattner et al., 1998). This simultaneous and reversible binding occurs because both ends of the MtCK octamer are identical and contain a series of exposed positively charged residues in their respective C-terminal regions (Schlattner et al., 1998). These contact sites have been shown to be enriched with the outer membrane protein porin, hexokinase, the inner membrane protein ANT and most importantly, octameric MtCK (Biermans et al., 1990; Kottke et al., 1991; Brdiczka & Wallimann, 1994; Beutner et al., 1998). This binding could provide the structural basis for the functional coupling of MtCK with ATP transport out of the matrix (through the adenine nucleotide translocase, or ANT) and for PCr transfer to the cytoplasm through the porin channels in the outer membrane. MtCK can therefore be thought to perform two important roles - (a) it comprises the "source" terminus for PCr formation in the IMS and (b) it plays a "quasi-structural"

Figure 2. MtCK is thought by many to play a dual role in the intermembrane space (IMS). The cartoon shown here serves to illustrate both the structural changes that may arise as a result of a shift in the octamer-dimer equilibrium and to highlight the organizational changes in vectorial transport of both the adenine nucleotides and the creatine/phosphocreatine pools as these changes occur. Octameric creatine kinase has been reported to form a complex with the inner membrane protein adenine nucleotide translocator (ANT) and the outer membrane protein porin (and hexokinase from the cytoplasm) forming contact sites which may act to inhibit the formation of the so-called permeability transition pore (PTP), thereby preventing mitochondrial-dependent apoptosis (Rojo et al., 1991).

9 role in contact site formation. Khuchua et al. (1998) have demonstrated using site- directed mutagenesis that destabilization of the octamer in vivo “profoundly reduced membrane binding” and, “markedly impacted the Cr - stimulated respiration” in rat neonatal myocytes, providing strong evidence that quaternary structure is directly related to physiological function in CK isoforms. Furthermore, Speer et al. (2005) have recently shown using transgenic mice that expression of MtCK in liver cells (which do not constitutively express CK) results in increased contact sites in mitochondria and enhanced resistance to detergent lysis. This strongly suggests that MtCK octamers have a structure-stabilizing role within mitochondria in addition to the clear role of CK in cellular energetics. The body of evidence indicating that MtCK’s octameric quaternary structure is intimately and perhaps even inextricably involved in the maintenance of mitochondrial membrane structure and in energy transport out of the mitochondria has attracted considerable attention. While a significant base of information centered on the MtCK - membrane interaction exists, the majority of attention has been focused on the nature and degree of the stability of the octamer itself (Gross et al., 1994; Gross & Wallimann, 1995; Koufen et al., 1999; Soboll et al., 1999; Eder et al., 2000; Schlattner et al., 2000; Schlattner & Wallimann, 2000; Pineda & Ellington, 2001; Wendt et al., 2003; Hoffman & Ellington, 2005). SarMtCK appears to form somewhat less stable octamers than UbiMtCK (Schlattner & Wallimann, 2000), and octamers of both rapidly dissociate into dimers upon conversion into the transition state analog complex (TSAC) that forms when these CKs are incubated with creatine, nitrate, and MgADP. This is thought to indicate that the conformational movements associated with the formation of the closed state that is believed to accompany substrate binding in phosphagen kinases (Forstner et al., 1998; Zhou et al., 2000; Lahiri et al., 2002; Yousef et al., 2003) interfere with dimer-dimer interactions (Schlattner & Wallimann, 2000). Our lab is engaged in an ongoing effort to understand both the nature and the consequences of the gene duplication events (and the subsequent divergence) that have occurred in the CK (and AK) genes over the one billion years or so (Nikoh et al., 1997) since the Porifera last shared a common ancestor with higher metazoans. One major focus in this effort has been designed to characterize the properties of primitive forms of

10 mitochondrial CK in an effort to more fully understand the structural changes which have led to the unique properties we now see in mammalian multimers built from Sar- and UbiMtCK subunits. This dissertation addresses the issue of octamer stability in an early mitochondrial creatine kinase using a comparative approach. Here we report the cloning, over- expression, and purification to homogeneity as a recombinant protein of a mitochondrial CK from the marine polychaete worm Chaetopterus variopedatus (referred to herein as CVMtCK) and a sarcomere-specific mitochondrial CK isoform from chicken cardiac tissue (SarMtCK), organisms which last shared a common ancestor at least 670 million years ago when deuterostomes diverged from protostomes (Doolittle et al., 1996). There is an extensive body of literature focused on both chicken sarcomeric and human ubiquitous MtCK- including X-ray crystal structures of both isoforms (Fritz-Wolf et al., 1996; Eder et al., 2000) - that has provided a comprehensive overview of MtCK octamer stability in birds and man. These data, in combination with our studies of the recombinant CVMtCK construct, have allowed us to show that CVMtCK octamers display the same disassociation and reassociation profiles under various temperature and concentration regimes as their mammalian counterparts but the rate of change in both directions is much more rapid than in the Sar- and UbiMtCK isoforms. Interestingly and perhaps more importantly, this CVMtCK construct is, at the same time, much more stable when converted to the TSAC. We have also addressed the role that certain specific residues located in the dimer-dimer interface may play in the relationship between structural differences and physiological function in this important family of homeostatic enzymes. Taken together, these observations indicate that there are significant differences in the stability of mitochondrial octamers of MtCK in polychaetes when compared to these same isoforms in birds and man, at least when measured in vitro, and that these the differences may be indicative of important functional differences within the mitochondria of living cells.

11 MATERIALS AND METHODS

RNA isolation

Total RNA from C. variopedatus had been previously isolated and used to clone and sequence the cDNAs for cytoplasmic CK and MtCK (Pineda & Ellington, 1999). Chicken cardiac muscle total RNA was isolated from heart tissue from a chick cadaver produced as a result of a sanctioned protocol in another research group. Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA). All chemicals were of reagent grade quality.

Preparation of full length cDNAs and expression constructs

Oligo dT-primed cDNA for both Chaetopterus and chicken RNA was prepared using Ready-To-GO-You-Prime First-Strand beads (GE Healthcare, Piscataway, NJ) and a lock-docking oligo dT primer (Borson et al., 1992). Full length cDNAs containing START and STOP codons for the mature MtCKs (minus the N-terminal mitochondrial targeting sequence) were generated by PCR amplification of the RT product (CVMtCK forward primer: 5’ ATGCGTTTAGGCACATCAAAGTC; CVMtCK reverse: 5’ TTAT- TTAGCAATGACATCATCAAT; SarMtCK forward: 5' ATGACTGTACATGAGAAG- CCGAAGCTC; SarMtCK reverse: 5' TCATTTCCTGCCAAAGTGGCAAAT). Ex Taq DNA polymerase (Takara, Madison WI) was used in the amplifications followed by brief treatment (72° C for 20 min) with Taq DNA polymerase (Promega, Madison, WI) to add d(A) overhangs on the cDNA. The PCR products were purified using Wizard PCR preps (Promega) and ligated into the pETBlue1 vector (Novagen -EMD Bioscience, Madison, WI) per the manufacturer's instructions. The construct was then transfected into NovaBlue cells (Novagen) and subjected to blue/white screening. Plasmids from positive clones were isolated, subjected to a restriction digest to ascertain insert size and orientation and then sequenced. Tuner (DE3) pLacI competent E. coli cells (Novagen) were transformed using plasmid constructs which had been verified as to correct

12 sequence and orientation of the inserts. It is these Tuner (DE3) cells that were used in the expression protocols. Glycerol stocks were prepared for transformed cells which had been validated as producing a CK product and these stocks were stored at -70 oC (see Fig. 3 for overview).

Expression and purification of recombinant proteins

SarMtCK was expressed in 1 liter of LB media by induction of the T7 promoter with isopropyl--D-thiogalactopyranoside (IPTG) at a final concentration of 0.84 mM during log phase and expression was permitted to continue for 6 h at 37 oC as was previously reported (Furter et al., 1992). CVMtCK expression level was subjected to an extensive optimization protocol after it was demonstrated using SDS/PAGE that the recombinant protein was expressed under control of the IPTG - induced T7 promoter. The recom-binant protein was expressed in an insoluble form (inclusion bodies) when expressed at 37 °C for 6 h. To determine conditions that would promote the expression of active and soluble CVMtCK, both the growth media (3 media; LB, M9, and 2XYT; and +/- glucose) and the growth conditions (expression temperature, growth phase at induction, time post-induction, and IPTG concentration) were systematically varied and CK activity in the soluble fraction was monitored. In the final conditions for CVMtCK expression, cells were grown to log phase at 37 oC in LB media, the incubation temperature was changed to 15 oC and the culture was allowed to equilibrate to the new temperature for 40 min before induction with IPTG (final concentration = 0.84 mM). Cells were permitted to express CVMtCK in the culture for 24 h at 15 oC to avoid driving the nascent protein into insoluble inclusion bodies. Cells containing both Sar- and CVMtCK constructs were harvested by centrifugation and stored at -70 °C for less then 1 week prior to lysis and purification. Both recombinants were purified to near homogeneity in two chromatographic steps using Blue Sepharose 6 Fast Flow affinity media (GE Healthcare) followed by cation exchange on Carboxymethyl (CM) Sepharose CL-6B media (GE Healthcare). All buffers were adjusted to pH at room temperature before chilling to 4 °C. Glycerol was

Figure 3. This flow chart illustrates the steps involved in the cloning and expression of the cDNAs coding for the mature CV- and SarMtCk proteins. (1) Total RNA was extracted from frozen (-70 °C) tissue using the method of Chomczynski and Sacchi (1987). (2) cDNA was reverse transcribed using a lock-docking oligo d(T) primer (Borson et al., 1992). (3) Full-length cDNAs coding for the mature proteins (mitochondrial-targeting sequence removed) were amplified from the RNA / DNA hybrid molecules using a gene-specific forward primer with a START codon added and a gene- specific reverse primer that included the STOP codon. (4) A 3' d(A) overhang was added by incubating the PCR product in TAQ polymerase allowing the full length cDNA to be ligated (5) into linearized pETBlue 1 vectors containing 3' d(U) overhangs. (6) The vector used to transform NovaBlue cells (Novagen) and these cells were subjected to blue / white screening to identify cells containing our plasmid. (7) Transformed cells were cultured and plasmid DNA was isolated using Qiagen mini-prep kits. This plasmid DNA was subjected to restriction digests to identify plasmids with the insert in the proper orientation relative to the inducible promoter. (8) Sequence-verified plasmids were transformed into Tuner (DE-3) cells (Novagen) and checked for expression of the MtCK insert. These tranformants were stored as glycerol stock solutions at - 70 °C.

14 5’ AAA(n) 3’ RNA (1) TTT(n) LD/d(T) RT

START RNA AAA(n) (2) TTT(n) cDNA

STOP PCR

START Mature (3) STOP CDS

TAQ

STOP-A Add 3’ (4) A-START d-ATPs

Ligase

Ligate into -A- -U- pETBlue-1 (5) -A- -U- Vector

Transform / Screen NovaBlue cells (6)

(7) Plasmid prep / Restriction Digest / Sequence

Transform Tuner (DE-3) pLacI cells (T-7 lac gene) (8)

15 added to 5% (W/V) for all CVMtCK buffers unless otherwise noted. Bacterial cell pellets were thawed and resuspended in ~ 60 ml of lysis buffer (CVMtCK lysis buffer: 50 mM Tris/HCl [pH 8.0], 150 mM NaCl, 5 mM EDTA, and 14 mM -mercaptoethanol [- MSH] in 10% glycerol; SarMtCK lysis buffer: 50 mM Tris/HCl [pH 8.0], 300 mM NaCl, 5 mM EDTA and 14 mM -MSH). Pellets were briefly homogenized using a Polytron (Brinkman, Westbury, NY), and the cells were lysed using 100 cycles of micro- fluidization (Microfluidics, Newton, MA) under nitrogen gas. The resulting lysate was centrifuged at 12000 x g at 4 °C for 20 min and the supernatant was exhaustively dialyzed against Blue Sepharose running buffer [50 mM Na phosphate (pH 6.5), 1.0 mM MgCl2, 0.5 mM EDTA and 1.0 mM dithiothreitol (DTT)]. The dialysate was applied to an 88 ml Blue Sepharose column (GE Healthcare), washed, and eluted with 200 mM NaCl. Total protein coming off the column was monitored using an ISCO UV detector (Lincoln, NE) set to 280 nm. Active fractions (CK reverse assay, see below) were pooled and exhaustively dialyzed against CM running buffer [CVMtCK: 10mM Na phosphate (pH 6.5), 50 mM NaCl, 0.5 mM EDTA, 1.0 mM DTT; SarMtCK: 25 mM Na phosphate (pH 6.5), 25 mM NaCl, 0.5 mM EDTA and 1.0 mM DTT]. The dialysate was applied to a 40 ml CM column (GE Healthcare), washed with running buffer, and eluted with a 400 ml, 0 -1.0 M linear NaCl gradient. UV (280) was monitored as above. Active fractions were checked for purity using SDS/PAGE (Laemmli, 1970), pooled, and protein concentration determined using a standard BioRad (Hercules, CA) protein assay. Purified recombinant proteins were concentrated using a 50 ml stirred ultrafiltration cell (Millipore, Bedford MA.) under pressure (using nitrogen gas) to ~5 mg/ml and stored at 4 °C in storage buffer containing 20 mM Tris/HCl (pH 8.0), 50 mM KCl, 0.5 mM EDTA and 1.0 mM DTT. A 1.5 mg/ml working stock solution was maintained in the same storage buffer.

Enzyme assays

Spectrophotometric CK assays were conducted at 25 oC in the reverse direction (phosphocreatine → MgATP) using a previously reported assay protocol (Strong &

16 Ellington, 1996)

Analytical procedures

Estimates of native molecular mass (Mr) values were conducted as previously described (Ellington et al., 1998). Octamer / dimer equilibria were determined by size exclusion chromatography using a pre-calibrated analytical Superdex 200HR size exclusion column (GE Healthcare) in Superdex running buffer [50 mM Na phosphate (pH 7.0), 150 mM NaCl, 0.5 mM EDTA and 1.0 mM DTT]. When required, MtCK stock preparations were diluted into buffer consisting of 20 mM Tris/HCl (pH 8.0 at room temperature), 50 mM KCl, 0.5 mM EDTA and 1.0 mM DTT. In routine experiments, a 20 to 50 µl aliquot of the MtCK dilution was injected into the Superdex column (flow = 0.4 ml x min-1) and elution was monitored for 60 min using a System Gold FPLC system (Beckman Coulter, Fullerton, CA). The relative proportion of octamers and dimers was evaluated by integrating the area under the relevant A (280) peaks using the image analysis program IMAGE (Scion Corporation, based on NIH IMAGE for McIntosh). Experiments evaluating the impact of protein concentration, temperature and conversion to the TSAC were conducted (additional details for each experiment are given in the figure legends). For dilution and temperature-induced dissociation of octamers, the resulting data were fitted to an exponential decay equation using the method of Kaldis et -1 al. (1994): At = Afinal + (A0 – Afinal) e (-kdis t) where At is the % octamer at time = t,

Afinal is the % octamer at equilibrium, A0 is the % octamer at time = 0, and kdis is the time constant for dissociation of octamers into dimers expressed as min-1. For rapid thermal transition studies it was possible to observe dimer association to octamer by raising the incubation temperature from 2 to 14 or from 2 to 20 oC. These preparations showed an exponential decrease in % dimer which reached equilibrium in approximately 2 h. Percent dimer data were fitted to the above equation (where A refers to % dimer) to yield -1 a time constant for dimer association (kassoc), also expressed as min . Oligomeric state was also evaluated by dynamic light scattering (DLS) using a Protein Solutions (Charlottesville, VA) DynaPro 99-D50 DLS instrument equipped with 824.6 nm 60 mW laser and a 45 µl micro sampler Peltier-cooled device using a 1.5 mm

17 quartz cuvette. All buffers were filtered through 0.22 µm syringe filters. Protein samples were centrifuged for 20 min at 13,000 x g at the appropriate incubation temperature in order to remove any particulates that would interfere with DLS measurements. The TSAC-induced octamer dissociation experiments were conducted by direct addition of 75 µl of 2X TSAC buffer to 75 µl of 4.0 mg/ml MtCK at time = 0 (final TSAC concentration: 50 mM NaNO3, 20 mM creatine, 5 mM MgCl, 4 mM ADP). Cuvettes were held in a refrigerated water bath at the set point temperature between measurements. Results were calculated as apparent hydrodynamic radius (Rh) and polydispersity (%PD; a measure of the size distribution of the particles) in the sample solution for each measurement.

Multiple Sequence Alignments

Sequence alignments were produced using CLUSTAL-X (Thompson et al., 1997) and visualized for print using GENEDOC (www.psc.edu/biomed/genedoc).

Homology Models Homology models were constructed using SWISS MODEL (Schwede et al., 2003). Briefly, appropriate quaternary structure templates for each protein were identified using a BLAST search of the ExNRL-3D database (extracted and annotated crystal structure information from PDB). Template and target sequences were aligned using CLUSTAL-X (Thompson et al., 1997). The target sequence and the quaternary structure template were loaded into SwissPDB Viewer (SPDBV) (Guex & Peitsch, 1997) and the target sequence was threaded onto the template after manually aligning the sequences in SPDBV based on the Clustal-X alignment. This PDB file was submitted via fttp to the Swiss Model automated protein homology modeling server (http://swissmodel.expasy.- org/swiss-model/). The resulting structure file was subjected to successive rounds of steepest descent energy minimization using GROMOS 96 (http://www.igc.ethz.ch- /gromos/) until the change in energy (∆E) between steps was less than 0.050 kJ/mol. Model validity statistics were determined using WHATCHECK (Hooft et al., 1996) and PROCHECK (Pontius et al., 1996), two open access protein structure analysis programs

18 that evaluate structural parameters ranging from bond lengths and torsion angles to advanced analysis of contacts and hydrogen bond networks. The structures were visualized using SWISS PDB Viewer (SPDBV; http://www.expasy.org/spdbv/) and an open-source version of PyMOL (DeLano Scientific LLC, San Carlos, CA).

Site Directed Mutagenesis

Analysis of aligned sequences for MtCK homologs reveals that a tryptophan (number 264 in chicken SarMtCK) appears to be conserved in all octameric MtCKs and mutation of this residue in the SarMtCK construct to a Cys severely disrupted the octamer (Gross et al., 1994). In order to more fully evaluate the role this residue plays in the octamerization of mitochondrial CK, we mutated the equivalent Trp of CVMtCK to Cys, Tyr [as found in the sponge Tethya, an obligate dimeric MtCK (Sona et al., 2004)], Leu, and the more conservative Phe. Mutations were performed on pETBlue 1 plasmid DNA containing the target construct using a Stratagene (La Jolla, CA) QuickChange II mutagenesis kit according to the manufacturer’s instructions (see Table 1 for a detailed list of the primers used). Briefly, the template plasmid DNA was amplified using 16 cycles of 95 °C for 30 sec, 56 °C for 30 sec, 68 °C for 16 min followed by a final annealing step of 7 min at 68 °C with PfuTurbo-HS DNA polymerase (Stratagene), dNTPs, a forward primer incorporating the desired base change, and the reverse compliment of the forward primer (See table 1 for a complete list of the primers used). The wild type plasmid was digested away using Dpn-1, a restriction enzyme with specificity for methylated DNA. The mutant constructs were used to transform XL-1 Blue Supercompetent cells (Stratagene) per the supplied instructions. Plasmids were collected from overnight cultures of the transformed cells and the insert was sequenced from both directions to verify the protein coding sequence. These verified plasmids were used to transform BL21 Tuner (DE3) cells (Novagen), then expressed and purified as described above. The purified mutants were subjected to DLS analysis where Rh and %PD were determined. Specific activity in the reverse direction (ATP production) for each mutant enzyme was also determined using the coupled reaction of Strong and Ellington (1996) and a standard BioRad protein assay.

19 Table 1. Sequences of the primers used for mutagenesis of the CVMtCK W264 residues showing wild-type residues and the Cys, Tyr, Leu, and Phe mutations as well as the melting temperature (Tm), nucleotide count (N), G and C base composition (% GC), and percentage of residues in the primer that are no longer complimentary to the wild type sequence (% mismatch) for each mutation.

CVMtCKW264 (wild type)

481 cgtggcatttggcataacgacaagaagaacttcttggtgtgggtgaatgaggaggaccac 161 R G I W H N D K K N F L V W V N E E D H

541 actcgtgtcatctccatgcagaagggtggcaacatgagggaagtgttcgatcgcttctgt 181 T R V I S M Q K G G N M R E V F D R F C

601 aacggcctgcaaaaggtggagaacttgatccagtctcggggcTGGgagttcatgtggaat 201 N G L Q K V E N L I Q S R G W E F M W N

661 gaacatttgggctacgttctgacctgccccagcaacctgggcactggtctgcgtgctggt 221 E H L G Y V L T C P S N L G T G L R A G

CV W264C

Target sequence: 5' gtctcggggcTGGgagttcatgtggaatgaacatttggg 3'

2AW264CF forward: 5' gtctcggggcTGCgagttcatgtggaatgaacatttggg 3'

2AW264CR reverse: 5' cccaaatgttcattccacatgaactcGCAgccccgagac 3'

Tm = 83.6 (N=39; %G/C = 53.8; % mismatch = 2.56)

CV W264Y

Target sequence: 5' gtctcggggcTGGgagttcatgtggaatgaacatttggg 3'

2AW264YF forward: 5' gtctcggggcTACgagttcatgtggaatgaacatttggg 3'

2AW264YR reverse: 5' cccaaatgttcattccacatgaactcGTAgccccgagac 3'

Tm = 79.9 (N=39; %GC = 55.3; %mismatch = 5.26)

CV W264L

Target sequence: 5' gtctcggggcTGGgagttcatgtggaatgaacatttggg 3'

CVW264LF forward: 5' gtctcggggcCTGgagttcatgtggaatgaacatttggg 3'

CVW264R reverse: 5' cccaaatgttcattccacatgaactcCAGgccccgagac 3'

Tm = 78.6 (N=39; %GC = 53.8; %mismatch = 7.69)

Table 1. Continued

CV W264F

Target sequence: 5' gtctcggggcTGGgagttcatgtggaatgaacatttggg 3'

CVW264FF forward: 5' gtctcggggcTTCgagttcatgtggaatgaacatttggg 3'

CVW264FR reverse: 5' cccaaatgttcattccacatgaactcGAAgccccgagac 3'

Tm = 77.5 (N=39; %GC = 51.3; %mismatch = 7.69)

21 RESULTS

The open reading frame coding for CVMtCK was described in work previously reported by our group (Pineda & Ellington, 1999). It consists of 1227 nucleotides coding for 409 amino acids. A 34 residue mitochondrial targeting sequence, located at the N- terminal end of the nascent protein, is cleaved yielding a mature protein consisting of 375 amino acids with an estimated pI of 9.70. Over-expression of CVMtCK in E. coli DE3 (TUNER) cells using a pETBlue 1 vector followed by extensive optimization of the expression and purification protocols resulted in routine yields of ~ 15 mg/L of essentially homogeneous CVMtCK recombinant protein. Figure 4 shows an SDS/PAGE of the DE3 (Tuner) cell lysate that demonstrates that our recombinant protein is expressed under control of the IPTG- induced promoter. The purification protocol was remarkably effective. Figure 5 shows the total UV absorbance overlaid on the the activity profiles of the eluted fractions for the low pressure chromatography on Blue Sepharose and CM Sepharose, respectively. Judicious pooling of the CM Sepharose fractions yielded an essentially homogeneous preparation (See lane 5 in Fig. 6). A final polishing step on Superdex 200PG (GE Healthcare) produced only minor enhancements in purity (lane 6, Fig. 6). In order to compare CVMtCK with a SarMtCK, we expressed and purified recombinant chicken SarMtCK using the same PetBlue1 (Novagen) system. Purification of this construct using published procedures (Furter et al., 1992) yielded ~5 mg of essentially homogeneous protein with minimal optimization. The relative molecular mass (Mr) for the denatured subunit of recombinant CVMtCK, as estimated using SDS/PAGE, was ~ 43 kDa, in good agreement with the Mr predicted from the primary sequence and with previous reports for CK subunit Mr values. Analytical size exclusion chromatography of the final recombinant preparation on Superdex 200HR FPLC system revealed the presence of both octamers and dimers (for a typical FPLC profile, see Fig. 7) with non-denatured Mr values of 347.3 and 61.5 kDa, respectively. Subunit and octamer Mr values of recombinant CVMtCK are virtually identical to values previously obtained for C. variopedatus MtCK purified from natural

Figure 4. SDS-PAGE of raw cell lysates from cell cultures started with 4 independent colonies of DE3 (Tuner) cells showing the high level of protein of the expected size from 15° C, 4 hour post-induction cultures as compared to cell cultures collected under the same conditions but without the IPTG inducer. Dashed arrow indicates the position of the 45 kDa band of ovalbumin. Solid arrows depict lanes corresponding to extracts of cells which were induced with IPTG.

Figure 5. UV (280 nm) and enzyme activity (CK reverse assay; MgATP production) plots generated from the Blue Sepharose and CM columns used to purify CVMtCK. (A) Raw cell lysate applied to the Blue Sepharose column and eluted with a single pulse of 150 mM NaCl. (B) Pooled, active Blue Sepharose fractions eluted from the CM column with a 400 ml, 0 - 1 M linear gradient of NaCl. Inserts show SDS/PAGE of active fractions from the respective stages.

Figure 6. SDS-Page of recombinant CVMtCK at various stages of the purification process. Lanes correspond following: Lane 1 and 7 - Mr markers (phosphorylase b, 97.4 kDa; BSA, (66.3 kDa); ovalbumin (45 kDa); carbonic anhydrase (31 kDa); soybean trypsin inhibitor (21.5 kDa); lysozyme (14.4 kDa). Lane 2 - raw un-induced cell lysate. Lane 3 - raw IPTG-induced cell lysate. Lane 4 - pooled active Blue Sepharose fractions. Lane 5 - pooled active CM fractions. Lane 6 - CM pool after Superdex 200PG size exclusion chromatography and concentration.

Figure 7. FPLC with a Superdex 200HR column demonstrates the relationship between octamers (left peak) and dimers (right peak). Here, the result of injecting 25 µL of 0.9 mg/ml (Bradford) purified protein is shown. Integration of the respective peaks yields the ratio of octamer to dimer in a given sample. Specific elution times (min) are indicated for each peak.

26 sources (Ellington et al., 1998). The measured Mr of 61.5 kDa for the CVMtCK dimer is consistent with measurements taken with the SarMtCK dimer and most likely reflects differences in the hydrodynamic properties of the “banana-shaped” dimer (seen also in the MtCK, MM and BB crystal structures) as compared to the more globular proteins used as standards (the dimer has an Mr of ~ 84 kDa when predicted from the open reading frame coding for the mature protein). To validate the interconversion of these two oligomeric forms of CVMtCK, the eluting octamer peak was collected, concentrated and reinjected yielding both octamers and dimers. The dimer peak from this latter run was collected, concentrated and reinjected yielding octamer and dimer peaks (Fig. 8). Clearly, there is reversible association of dimers to form octamers. To evaluate the biophysical parameters of the octamer-dimer equilibrium in CVMtCK and to compare this equilibrium with the well-characterized chicken SarMtCK isoform, the impacts of protein concentration, temperature and conversion to the TSAC were investigated. We initially used size-exclusion chromatography as was done in previously published work on human Ubi- and chicken SarMtCKs (Gross et al., 1994; (Gross et al., 1994; Gross & Wallimann, 1995; Eder et al., 2000; Schlattner et al., 2000) and later evaluated these parameters using dynamic light scattering. The octamer-dimer equilibrium for CVMtCK is highly dependent on protein concentration (Fig. 9) as the proportion of octamers dramatically increased from less than 20 % at 0.1 mg/ml to greater than 90 % as the CVMtCK concentration approached 1.5 mg/ml. A similar pattern has been observed for chicken SarMtCK (Gross & Wallimann, 1995) and both human Sar- and UbiMtCKs (Schlattner & Wallimann, 2000), all in the absence of TSAC components. The octamer-dimer equilibrium upon dilution of CVMtCK and the time constant for octamer dissociation (kdis) are highly temperature dependent. Both % octamer and kdis values increased from 2 °C to 28 °C and decreased at higher temperatures (Fig. 10) until becoming immeasurable at temperatures above 32 °C, most likely due to protein breakdown. When chicken SarMtCK (at 0.1 mg/ml) was incubated

Figure 8. Demonstration of the interconvertibility of the octameric and dimeric forms of CVMtCK. (A) Recombinant MtCK was injected into a Superdex 200HR column and a portion of the octamer peak was hand collected, concentrated, and stored at 4° C for 24 h. (B) The sample was then injected into the Superdex column and the well separated dimer peak was collected, concentrated, and again stored at 4° C for 24 h. (C) Injection of this sample revealed that octamers and dimers of CVMtCK are interconvertable.

Figure 9. The effect of protein concentration on the extent of octamerization (expressed as % octamer) for recombinant CVMtCK. Each value is a mean ± 1 S.D. (n = 3). A concentrated stock solution was diluted to the indicated concentrations and the samples were allowed to equilibrate at 4 °C for 24 h before each measurement.

Figure 10. The effect of temperature on the extent of octamerization (expressed as % octamer; left panel) and on the rate constant for octamer dissociation (kdis, expressed as min-1; right panel). Experiments were conducted at 2, 4, 8, 16, 20, 26, 28 and 32 °C by diluting 1.5 mg/ml CVMtCK to 0.1 mg/ml and monitoring the % octamer over a time course until the enzyme reached a new equilibrium.

30 at a range of temperatures from 0 to 30 oC, a virtually identical equilibrium of octamers and dimers vs. temperature curve was generated (Gross & Wallimann, 1995). Similarly, the rate of SarMtCK octamer dissociation increased dramatically as temperature was increased to 28 oC and then fell, in a pattern similar to that of CVMtCK (Gross & Wallimann, 1995). Although CVMtCK and chicken SarMtCK showed great similarity with respect to the impact of protein concentration and temperature on octamer stability, there were dramatic differences in the rate of octamer dissociation upon dilution. The time course of dilution-induced octamer dissociation for both recombinants was compared at 8 and 20

°C (Fig. 11). CVMtCK dissociated much more rapidly at both temperatures with kdis values of 1.90 x 10-2 min-1 and 1.41 x 10-2 min-1 at 20 and 8 °C, respectively, two orders of magnitude higher than than what was observed for SarMtCK (5.35 x 10-4 min-1 and 4.51 x 10-4 min-1 at 20 °C and 8 °C, respectively). Interestingly, both proteins reached a similar final equilibrium of octamers and dimers of 65 – 70 % octamer at 20 °C and 40 – 50 % octamer at 8 °C, in spite of the significant differences in dissociation rates (Fig. 11). Table 2 shows a summary of the octamer dissociation results with half life estimates

(calculated as τ1/2 = ln 2/k as in Kaldis et al., 1994) based on the observed decay constants. The half life for SarMtCK upon dilution to 0.1 mg/ml was >30 times longer than that of CVMtCK. Note that addition of 5 % glycerol to buffers in the SarMtCK experiments had no impact on octamer stability (data not shown). Since CVMtCK dissociates into dimers with a rapid time course and the final equilibrium is highly temperature dependent, it was possible to conduct thermal jump studies in which dimer association into octamer and octamer dissociation into dimer could be observed using the same protein dilution. Two thermal regimes were utilized at a protein concentration of 0.1 mg/ml (approximately 1 µM dimer and 0.18 µM octamer at 2 oC) – a thermal jump from 2 to 14 °C (dimer association), then a transfer from 14 to 2 °C (octamer dissociation) and a thermal jump from 2 to 20 °C, followed by a transfer from 20 to 2 °C. When CVMtCK was fully equilibrated to 2 °C and the temperature was

changed to 14 °C, the % octamer increased from 11.7 % to 58.9 % with a kassoc value of 5.01 x 10-2 · min-1 (Fig. 12 and Table 2). When the same CVMtCK dilution was transferred back to 2 °C there was a rapid dissociation yielding 12 % octamer at

Figure 11. Comparison of the time course of dilution-induced octamer dissociation into dimers for recombinant SarMtCK (left panel) and CVMtCK (right panel). Recombinant proteins were diluted from 1.5 mg/ml to 0.1 mg/ml at two temperatures 8 and 20 °C and the extant of octamerization (expressed as % octamer) was monitored over time. Values for kdis are indicated for each treatment.

Figure 12. Thermal jump studies of the dilution - induced dissociation into dimers and reassociation of dimers back into octamers in recombinant CVMtCK. Two regimes were used: 2 to 14 °C and 2 to 20 °C. Each time point value is a mean +/- 1 S.D. (n=3). Returning the protein to 2 °C resulted in a rapid dissociation yielding 11.7 % octamer at -2 -1 equilibrium with a kdis value of 1.83 x 10 x min . These results show that octamer formation (dimer association) is rapid and is accelerated by increased temperature.

Table 2. These data demonstrate the impact of temperature on the octamer content at equilibrium, the time constant for octamer dissociation (kdis), and the octamer half-life when CVMtCK and SarMtCK were diluted to 0.1 mg/ml and monitored over time at the indicated temperatures.

-1 Temp (°C) % Octamer kdis (min ) 1/2 life (min)

CVMtCK 2 9.7 4.34x10-3 159.7 CVMtCK 4 19.5 5.57x10-3 124.4 CVMtCK 6 27.3 9.00x10-3 77.0 CVMtCK 8 39.4 1.41x10-2 49.2 CVMtCK 14 47.0 1.50x10-2 46.2 CVMtCK 20 64.6 1.90x10-2 36.5 CVMtCK 26 68.2 2.14x10-2 32.4 CVMtCK 28 72.2 2.26x10-2 30.7 CVMtCK 30 71.2 2.00x10-2 34.7 CVMtCK 32 56.8 1.14x10-2 60.8 SarMtCK 8 48.0 4.51x10-4 1536.9 SarMtCK 20 72.1 5.35x10-4 1295.6

34 -2 -1 equilibrium with a kdis value of 1.80 x 10 · min . Similarly, when the temperature was changed from 2 to 20 °C the proportion of octamers increased from 11.7 % to 66.3 % -2 -1 with a kassoc value of 7.98 x 10 · min . Returning the protein to 2 °C resulted in a rapid -2 dissociation yielding 11.7 % octamer at equilibrium with a kdis value of 1.83 x 10 · min-1. These results show that octamer formation (dimer association) is rapid and is accelerated by increased temperature. In order to compare CVMtCK to the previously characterized SarMtCK (Gross & Wallimann, 1995), both recombinant proteins (at greater than 1.5 mg/ml and 4 °C) were converted to the transition state analog complex (TSAC) by exhaustive dialysis against Superdex running buffer containing 20 mM creatine, 5 mM Mg2+, 4 mM ADP, and 50 mM NaNO3. In contrast to the observed results on octamer stability at low protein concentrations in the apo state, when converted to the TSAC, the percentage of CVMtCK existing as octamers fell from ~ 95 % at time = 0 to only ~ 88 % after 24 h and attained an apparent equilibrium within 3 d at 84.5 % octamer while SarMtCK fell from ~95 % octamer to 19 % octamer in only 24 h and appeared to completely dissociate into dimers after 48 h as measured using FPLC (Fig.13, top). Given the rapid time course of CVMtCK octamer - dimer interconversion in the thermal jump studies noted above, it was possible that the observed stability of the CVMtCK octamers in the TSAC was an artifact of our procedure and the apparent stability was actually due to the loss of the TSAC components as a result of dilution in the Superdex 200HR column running buffer during the run. This would be most pronounced if the TSAC components where removed from the active sites while the protein itself remained at concentrations high enough to avoid octamer dissociation. These conditions would lead to the concentration - driven reassociation of the TSAC- induced dimers into octamers and it would appear that CVMtCK did not dissociate into dimers in the TSAC when in fact the protein had simply been converted back to the apo state. CVMtCK (at 2.5 mg/ml, a concentration known to yield a % octamer content > 95 %) were analyzed. There was a hyperbolic relationship between apparent hydrodynamic radii (Rh) and actual Mr values; apparent Rh values for the MM-

Figure 13. Comparison of the effect of conversion to the transition state analog complex on SAR- and CVMtCK using FPLC and DLS. (A) Protein at 1.5 mg/ml was dialyzed against TSAC components, incubated at 4 °C and aliquots were removed with time for assessment of octamer content using FPLC. (B) 4.0 mg/ml samples of both recombinant proteins were diluted to 2.0 mg/ml with 2X TSAC buffer at T=0 and incubated at 4° C. Apparent hydrodynamic radius (Rh) was monitored over 7 d using dynamic light scattering (DLS). Data shown are for the first 3 d, well after a new equilibrium was reached. Pre-conversion values are indicated (*). Plotted values represent a mean +/- 1 S.D. (n = 3) and final [TSAC] = 50 mM NaNO3, 20 mM creatine, 5 mM MgCl2, 4 mM ADP.

36 CK dimer and CVMtCK octamer were 4.06 +/-0.054 nm and 8.02 +/-0.054 nm (mean +/- 1 S.D., n=3) respectively (Fig. 14). This indicates DLS system clearly provides sufficient resolution to be able to discriminate between dimeric and octameric forms of CK. To further understand the relationship between Rh as measured on the DLS and the ratio of octameric to dimeric CVMtCK, we prepared a series of proportional mixtures (by mass) of CVMtCK and MMCK, again taking care to maintain a final CVMTCK concentration well above that known to drive the octamer : dimer ratio very strongly towards the octameric form. Once again, we saw an Rh of ~ 4.0 nm for the pure dimer and ~ 8.0 nm for the nearly-pure octamers. The proportional mixtures of octamers and dimers, ranging from 10 to 90 % octamer, exhibited a hyperbolic relationship (Fig. 15) again indicating that this method can indeed be used to discriminate between octameric and dimeric molecules in solution, although the precise determination of % octamer may be somewhat limited in mixed populations, especially at higher % octamer values. In order to confirm the ability of our DLS techniques to determine the effect of dilution over the run time for the FPLC-determined measurements, we prepared three concentrations of CVMtCK and monitored the effect of dilution over a time course (Fig. 16). Here, the effect was to shift the FPLC - determined concentration - dependency curve to the right with no significant change in the overall trend or time course for dissociation of the octamers. In total, these experiments demonstrated the DLS method to be a valid alternative to studies conducted using FPLC (a representative DLS scan can be seen in Fig.17). To summarize previously described results, the FPLC-measured conversion of the CVMtCK recombinant protein at 4 oC to the TSAC (protein concentration was at least 1.5 mg/ml) resulted in minimal changes in oligomeric state over the seven 7 d observation period. In contrast, SarMtCK rapidly dissociated into dimers as evidenced by a time dependent decrease in the % octamer values, reflecting a mixture of octamers and dimers, until a stable value was attained closely corresponding to that of a dimer. These FPLC-based TSAC-induced dimerization experiments were repeated, this time monitoring Rh on the validated DLS, and again the oligomeric state of recombinant CVMtCK was not significantly changed, while SarMtCK dissociated into dimers almost completely within 24 h. Thus, we have clearly shown by two independent methods (size exclusion

Figure 14. Standard curve generated by plotting apparent hydrodynamic radius (Rh) of a series of protein standards vs. molecular mass as determined by dynamic light scattering at 4 °C and 2.5 mg/ml (rabbit muscle CK = 2.0 mg/ml). The position of dimeric rabbit muscle (MMCK) and octameric CVMtCK are indicated (13.7 kDa: ribonuclease-A, 25.0 kDa: Chymotrypsin, 43.0 kDa: ovalalbumin, 85.9 kDa: MMCK, 158.5 kDa: aldolase, 232.0 kDa: catalase, 347.0: CVMtCK, 440.0: ferritin). Each value is a mean +/- 1 S.D. (n=3) and Mr is expressed as kDa.

Figure 15. Curve generated by plotting apparent hydrodynamic radius vs. % octamer (by mass) as determined by dynamic light scattering at 4 °C. Octameric CVMtCK (final concentration = 2.0 mg/ml) was combined with dimeric rabbit MMCK in the indicated proportions. Each data point is a mean +/- 1 S.D. (n = 3).

Figure 16. Determination of the final octamer/dimer equilibrium state of CVMtCK at three protein concentrations. After incubating overnight at 20 °C, the samples were transferred to the DLS and the microsampler temperature was changed to 2 °C (T = 0). Apparent hydrodynamic radius was monitored until a new equilibrium was reached. Hydrodynamic radius (Rh) at 20 °C is indicated. Plotted values represent a mean +/- 1 S.D. (n = 3).

Figure 17. This figure is representative of DLS data from a 2.0 mg/ml sample of CVMtCK at 20 °C showing a monomodal octamer species with a hydrodynamic radius (Rh) of 8.02 and a polydispersity (%PD) of 19.5. 100 % of the sample mass is represented within this curve.

41 chromatography and DLS) that the CVMtCK octamer is intrinsically more stable as a TSAC than SarMtCK. This suggests that differences may exist in the nature of the conformational movements occurring during substrate binding that affect amino acids taking part in interactions acting across the dimer – dimer interface and these differences may act to stabilize the CVMtCK octamer in contrast to the rapid and virtually complete dissociation of SarMtCK octamers under identical conditions. Multiple sequence alignment (Fig. 18) revealed that CV- , Sar- , and UbiMtCKs exhibit a high degree of sequence conservation. Sequence comparisons using the BLOSSUM 62 algorithm in CLUSTAL (Thompson et al., 1997) showed that the amino acid sequence of CVMtCK is 64 % identical and 78 % similar to that of chick SarMtCK and 67 % identical and 79 % similar to that of human UbiMtCK in spite of the fact that they last shared a common ancestor at least 550 million years ago (Pineda & Ellington, 1999). Note that chick Sar- and human UbiMtCKs display 78 % identity and 89 % similarity (Table 3). In order to integrate our experimental observations of octamer stability with structural factors at the dimer – dimer interface and at other places within the molecule, we used available PDB file crystal structures of Sar- (PDB: 1CRK) and UbiMtCK (PDB: 1QK1) as templates to construct a CVMtCK homology model. Figure 19A shows an image of the entire CVMtCK octamer (with all the side chains) looking through the central pore. This model shows a cuboidal octameric structure with dimensions of 114.52 Ǻ x 119.70 Ǻ. (Sar- and UbiMtCK are 115.50 Ǻ x 100.44 Ǻ and 108.19 Ǻ x 84.28 Ǻ, respectively). All three have a central pore of approximately 20 Ǻ. The highly disordered N-terminal residues are clearly visible within the pore where they interact and contribute to octamer stability. Figure 19(B) shows a view of a single CVMtCK dimer, this view is orthogonal to the octamer shown in figure 19(A). Again, the N-terminal residues that have been implicated in octamer stability are clearly seen, as is the classic “banana- shaped” dimer that is characteristic of all CK dimers described to date. This shape was invoked earlier in order to explain the unexpectedly long FPLC retention time when CVMtCK dimers were compared to a set of globular protein standards during molecular mass determinations.

Figure 18. Multiple sequence alignment showing identical residues shared between CVMtCK (accession #AAK35006), chicken SarMtCK (accession #P11009), and human SarMtCK (accession #P12532) . Absolutely conserved residues are indicated in red, residues conserved in two of the isoenzymes are indicated in gray. Consensus sequence is listed below the alignment (1: DN; 2: EQ; 3; ST; 4: KR; 5: FYW; 6: L I V M).

Table 3. Percent identity and similarity scores for the amino acid sequences of CVMtCK, chicken SarMtCK and human UbiMtCK.

Chicken SarMtCK Human UbiMtCK

CVMtCK Identity 64 % Identity 67 % Similarity 78 % Similarity 79 %

Human UbiMtCK Identity 78 % Similarity 89 %

Figure 19. All atom CVMtCK homology model showing (A); a top view of the octamer (through the 20 Ǻ central pore) and (B); two monomers (view is orthogonal to that in A) covalently bonded into a single dimer, the basic unit of MtCK quaternary structure. Images are constructed in PDB and visualized in PyMol.

45 Analysis and comparison of a suite of stereochemical parameters from the CV- homology model and the UbiMtCK crystal structure using two independent validation suites, PROCHECK (Pontius et al., 1996) and WHATCHECK (Hooft et al., 1996) suggested that our model was stereochemically realistic. This can most easily be seen in the Ramachandran plots generated during PROCHECK runs as shown in Figure 20. Both model and template are composed of over 3000 residues and in this plot of Phi – Psi angles, 88.8 % of the residues in the Ubi- structure are found in the most favored regions of the plots while 88.2 % of the CVMtCK residues are found in these same regions (Table 4). It should be noted here that even this small discrepancy between model and template disappeared when the first five N-terminal residues of the CV model were removed prior to generation of the Ramachandran plot (data not shown). This occurs because our model retained residues not resolved in the crystal structures due to the high B-factor (an indicator of kinetic energy) inherent to flexible loop regions which precluded resolution using X-ray crystallography. In the published structures, these regions involve the N- and C- terminal residues as well as the flexible loop region (number 101 – 110) in the SarMtCK numbering scheme). This can most easily be seen in Figure 21 where residues with no correspondence between model and template are shown in red. This figure clearly shows the N-terminal amino acids within the central pore that are unique to our model and allows one to visualize the C-terminal residues unique to UbiMtCK. It is also possible to visualize the red-colored residues of the flexible loop region in Figure 21 by carefully investigating the residues near the cleft between the large and small domains. In spite of these differences, our all - residue Ramachandran plots indicated that only ~ 0.2 % of the amino acids in our model and in the UbiMtCK crystal structure were found in disallowed regions (4 residues in Ubi- and 5 in CVMtCK). All other parameters, such as Chi-1 / Chi-2 plots, torsion angles, bond lengths, and more esoteric validation parameters such as hydrogen bond network energies and hydrophobicity (or “in – out” values) we investigated have shown a similar degree of concordance across the model and crystal structures. Another indication of model validity can be arrived at by calculating the total energy contained within the structures (Table 5). This method provides an absolute measure of model quality without relying on relative scores. Here the total energy as

Figure 20. Ramachandran plots (Psi/Phi plots) of the backbone molecules in the CVMtCK homology model (top) and the UbiMtCK structure (bottom) that was used as a template for modeling. These plots indicate the most stable conformations for the N – Cα (or Phi) and Cα – C (or Psi) torsion angles in the backbone atoms of a polypeptide. Combinations of Phi / Psi angles that will result in collisions are therefore sterically “disallowed” in a peptide chain. These disallowed residues are circled.

Table 4. Ramachandran plot statistics for the CVMtCK homology model and the UbiMtCK PDB entry that was used as a template (see Fig. 21 for the Ramachandran plots).

CVMtCK UbiMtCK

Residues in most favored regions 2271 88.2 % 2331 88.8 %

Residues in additional allowed regions 289 11.2 % 255 9.7 % Residues in generously allowed regions 12 0.5 % 33 1.3 %

Residues in disallowed regions 4 0.2 % 5 0.2 %

Non-glycine / non-proline residues 2576 100.0 % 2624 100.0 %

End residues (excl. Gly, Pro) 16 16

Glycine residues (shown as triangles) 256 240 Proline residues 152 152

Total residues 3000 3032

Figure 21. Backbone (N, Cα, C) atoms of the CVMtCK homology model superimposed onto the UbiMtCK template molecule and colored by RMS deviation from the template structure. Top view shows the CV octamer looking “down” through the central pore, and the image on the bottom shows the molecule rotated 90°. To create this image, both PDB files were loaded into SPDBV, superimposed, and a structural alignment was generated. Each backbone atom was then colored according to its RMS deviation from the corresponding amino acid of the UbiMtCK structure. Dark blue indicates good superposition whereas red indicates zero correspondence. Intermediate fit is indicated by yellow and green according to the natural spectrum of white light. Note the N- terminal amino acids (red in the center of the left image) with no correspondence to residues in the UbiMtCK structure and and the C-terminal residues (red in the top and bottom of the right image) with no corresponding residues in the CVMtCK molecule. Residues of the flexible loop (101 – 119 in CVMtCK) can be seen, especially in Figure 21A near the cleft in the upper right of the figure. As noted in the text, these loop residues are residues not well resolved in the Ubi –crystal structure.

Table 5. Comparative validation statistics for the CVMtCK octameric homology model showing the calculated energy in the model and in the two octameric crystal structures reported to date. Also shown is root-mean-square deviation (RMSD; an indication of the “best fit” between two molecules) for the Cα (a) and the backbone atoms (b) of CVMtCK as compared to the UbiMtCK and SarMtCK X-ray structures. The very low rms deviation between UbiMtCK and the model is indicative as its use as the template and the subsequent rounds of energy minimization carried out on CVMtCK during model optimization. Note that the RMSD values for the model against SarMtCK are nearly identical to the RMSD values between the crystal structures themselves.

UbiMtCK SarMtCK Energy (kJ·mol-1) RMSD (Ǻ) RMSD (Ǻ)

CVMtCK -155.56 0.13(a) / 0.16(b) 1.30(a) / 1.31(b)

UbiMtCK -137.63 - 1.32(a) / 1.33(b)

SarMtCK -115.74 - -

calculated by GROMOS96 (http://www.igc.ethz.ch/gromos/) is - 155.56 kJ/mol for CV, -137.63 kJ/mol for Ubi-, and -155.74 kJ/mol for SarMtCK. These values are within 20 % of each other and again suggest that our model is a reasonable representation of the CVMtCK molecule, at least in a stereochemical sense. There is one more parameter of model quality that should be discussed here because it provides a direct comparison between the backbone atoms (N, Cα, and C) that make-up the peptide chains within the proteins. This is the Root Mean Square Deviation, or RMSD. RMSD values indicate how far these backbone atoms vary from complete correspondence with one another in 3-dimensional space. In other words, if one were to compare a protein structure to itself, the calculated RMSD value would be equal to zero. Here, we found it more useful to compare our CV- model and the Ubi- template structure to the SarMtCK molecule. This analysis revealed an RMS deviation of 1.31 Ǻ between CV- and SarMtCK and 1.33 Ǻ between CV- and the Ubi- structure when we evaluated the backbone superimposition (Table 5). These values are within the limits of resolution for the crystal structures themselves. To summarize, the model validation analysis software programs WHATCHECK and PROCHECK, which analyze a variety of parameters from simple bond lengths and torsion angles to more complex networks of interactions such as hydrogen bonds and hydrophobicity, indicate that our model does not contain any forbidden interactions and that for all parameters, the results are not significantly different from the values obtained when the crystal structures of Ubi- and SarMtCK were analyzed using identical tests. These statistics indicated that we could be confident that our model structure was a reasonable representation of the CVMtCK molecule given certain limitations inherent to the modeling technique itself. The most serious of these limitations arises from the fact that the template crystal structure with the highest sequence homology and identity level (UbiMtCK) has been resolved to only 2.0 Ǻ. This clearly represents an absolute limit that even a perfect model could be expected to exhibit and is at a level of resolution that is of the same order as the interactions that we were trying to understand. Using careful analysis of sequence alignments and by comparing our homology

52 model with the published crystal structures, we were able to identify a number of residues that appear to be involved in octamer stability. These were identified by searching the model structure for residues within one particular dimer that are located within a specific distance (3.2 Ǻ) of a residue on an adjacent dimer with which it is capable of having an interaction. These potential interactions include salt bridges, hydrogen bonds (HB), hydrophobic and polar interactions, and straightforward van der Waals forces. In order to visualize these differences in the dimer – dimer interactions among the three mitochondrial CK octamers, we created a surface rendition of a single dimer from each of the 3 isoforms using PyMOL and annotated the interacting residues using red for salt bridges and green for polar interactions and hydrogen bonds (Fig. 22). Using the above criteria, we identified 3 salt bridges, 6 sets of polar interactions and 4 potential hydrogen bond pairs acting across the dimer – dimer interface in CVMtCK. As can be seen in Table 6, the salt bridges consist of two Arg – Glu pairs (R151 – E148; E155 – R151) coordinated by helices (residues 143 - 158 in SarMtCK) within the large domains of monomer “A” (of dimer A/E) and monomer “H” (of dimer D/H) and another pair (E155 – R151) within monomer “E” (of dimer A/E) and monomer “C” (of dimer C/G). We have also identified 6 distinct regions of polar interactions involving N-terminal residues (small domain) across the interface, and 4 potential sets of hydrogen bonds, again involving N-terminal residues. One of these polar interactions is between G263 and S12. Interestingly, with the exception of a potential hydrogen bond pair between E145 and L7, the G263 – S12 interaction is the only definitive interdimer pair that acts across the large and small domains. This interaction pair becomes even more interesting when one realizes that G263 is directly adjacent to a tryptophan residue (W264 in SarMtCK) that is absolutely conserved in all mitochondrial octamers of CK described to date. Furthermore, this tryptophan has been directly implicated in maintenance of stability within the octamer by the ETH group (Gross et al., 1994; Gross & Wallimann, 1995), using site- directed mutagenesis and three different techniques to evaluate stability, FPLC, tryptophan quenching experiments, and DLS. By way of contrast, analysis of the UbiMtCK crystal structure indicated that the

Figure 22. Surface representations of human UbiMtCK (A), CVMtCK (B) and SarMtCK (C) showing residues located within 3.2 Ǻ of contiguous dimers. Residues participating in salt bridges are rendered in red; other interactions (polar and potential hydrogen bonds) are indicated in green.) Salt bridges between the 143 -158 alpha helices (1) and hydrophobic interactions involving Gly263 / Trp264 with Ser11 / Pro31 (2) are indicated. Point of view in each image is indicated by the arrow in the "top" view of a representative octamer (D). Molecular surfaces are rendered using PyMol (Warren L. DeLano. "The PyMol Molecular Graphics System." DeLano Scientific LLC, San Carlos, CA USA. http://www.pymol.org).

Table 6. Residues of Ubi-, Sar-, and CVMtCK octamers at the dimer: dimer interface that are located within 3.2 Ǻ of an adjacent dimer. To construct this table, the octamer was conceptually bisected by a plane perpendicular to the central pore and visualized using SPDBV. Monomers above the plane were labeled A, B, C, and D, and monomers below the plane were labeled E, F, G, and H such that the dimers could be labeled A/E, B/F, C/G, and D/H. The small image directly below shows the two dimers (B/C has been omitted) that share an interface with A/E. Dimer A/E was then selected and all residues within 3.2 Ǻ of A/E were identified. The amino acid interaction pairs identified in this way have been listed here (hydrogen bonds are indicated as “HB”). Gray highlights indicate interactions that are unique to a particular isoform.

D A C

H E G

A H D G C Interaction CV R151 NH1 E148 OE2 Salt CV E155 OE1 R151 NH2 Salt CV G263 CO S12 OG Polar CV H6 ND R38 NH1 Polar CV L7 O Y9 N Polar CV Y9 NH L7 O Polar CV R38 O H6 NE2 Polar

E H D G C Interaction CV E155 OE2 R151 NH1 Salt CV S12 H G263 O Polar CV H6 NE2 R38 O HB CV L7 CO Y9 NH HB CV L7 CD E145 OE1 HB CV Y9 NH L7 CO HB

55 Table 6. Continued

A H D G C Interaction Sar K7 O K7 NG Polar Sar L8 L8 Polar Sar S12 OG G263 O Polar Sar G263 O S12 OG Polar Sar G263 P31 Polar

E H D G C Interaction Sar L8 L8 Polar Sar S12 OG G263 O Polar Sar P31 G263 Polar

A H D G C Interaction Ubi R6 NH1 D39 OD1 Salt Ubi R7 NE E145 OE1 Salt Ubi D39 OD1 R6 NE Salt Ubi E145 OE R7 NE Salt Ubi R151 NH1 E148 O2 Salt Ubi R7 NH2 A140 O Polar Ubi R7 O Y9 O Polar Ubi Y9 N R7 O Polar Ubi G263 S12 OG Polar

E H D G C Interaction Ubi R7 NE E145 OE Salt Ubi D39 OD1 R6 NH1 Salt Ubi E145 OE1 R7 NH1 Salt Ubi R151 NH1 E148 OE2 Salt Ubi D155 OD2 R151 NH1 Salt Ubi R6 NH1 Y9 O Polar Ubi Y9 O R6 NH2 Polar Ubi Y9 N R7 O Polar Ubi S12 OG G263 O Polar Ubi R7 O Y9 N Polar Ubi G263 O S12 OG Polar

56 dimer – dimer interactions suite consists of 11 salt bridges (3 salt bridges in CVMtCK), and 11 sets of weaker interactions (10 in the CV- model). For SarMtCK, we identified no salt bridges and 10 polar interactions (For a detailed list see Table 6). Our analysis of the Sar- and UbiMtCK dimer – dimer interactions is in good agreement with work published by Wallimann’s ETH group, the authors that originally reported the structures (Fritz- Wolf et al., 1996; Eder et al., 2000), indicating that our strategy for identifying these interactions in the CVMtCK octamer homology model was sound. This analysis revealed that there are significant differences in the nature of the inter-dimer interactions. This is especially pronounced when one considers the salt bridges acting across the dimer – dimer interface. Table 6 indicates that the relatively more stable octamers of Ubi- and CVMtCK are held together with salt bridges, polar interactions, and a small network of hydrogen bonding residue pairs while the less stable SarMtCK octamers are apparently coordinated without the use of salt bridges and stability, such as it is, is conferred by a series of polar interactions. While our structural analysis shows that a variety of interactions may stabilize the CVMtCK octamer, one residue, a tryptophan at position 264, is particularly interesting. This Trp264 appears to be conserved in all octameric MtCKs and mutation of this residue to a Cys in the SarMtCK construct severely disrupted the octamer (Gross et al., 1994). Additional evidence for the critical role of W264 in octamerization is found when one examines the equivalent residue in the MtCK from the sponge Tethya aurantia, the only MtCK that has been definitively demonstrated to exist as a dimer (Sona et al., 2004). In Tethya MtCK, this residue is a tyrosine. Interestingly, investigation of amino acids at this position in the multiple sequence alignment shown in detail in Figure 23 indicate that this residue is not a Trp in any of the very primitive isoforms known to date (it is a His, Tyr, or Asn in other sponges and a His in the cnidarian Hydractinia). Figure 24 shows the relative position of the CV W264 residue (which comparision shows is very similar to the orientation in the SarMtCK PDB file; data not shown) as compared to the equivalent Y264 in the sponge Tethya isoform (the Tethya data were generated by S. Sona) indicate that this residue is not a Trp in any of the very primitive isoforms known to date (it is a His, Tyr, or Asn in sponges and a His in the cnidarian Hydractinia). Figure 24 shows the

Figure 23. A ClustalW multiple sequence alignment of selected residues from mitochondrial isoforms found in organisms ranging from sponge to man. This alignment shows the Trp264 (SarMtCK numbering, arrow) residue that has been demonstrated later in this work to be essential for octamer stability as well as the Tyr, His and Asn substitutions (red) one would expect to destabilize the octamer. The Trp264 is absolutely conserved in all octameric MtCKs reported to date. In the dimeric Tethya (a poriferian) MtCK isoform it is a Tyr (Sona et al., 2004) and it is a Asn or a His in other primitive organisms. Notice the Arg / Lys to Asp substitution (red) at position 262 in the same organisms. These positively charged residues are also conserved, suggesting that Suberites (SuberMtCK - a sponge - unpublished data from our group) and Hydractinia (HyMtCK, a hydrozoan - assembled from EST contigs) MtCKs will be found to exist as dimers. It is interesting to note that Uda et al. (2005) have reported a mitochondrially targeted taurocyamine kinase from Arenicola (a marine annelid) that also shows the pattern of conservation associated with octamerization. [AphroMtCK (Aphrocallistes), a sponge – unpublished data from our group; softcoral (Dendroephthya) - from Suzuki (personnal communication); NeanthesMt (Neanthes) a polychaete and SpurpMtCK (Strongylocentrotus), a sea urchin - from GenBank; CionaMtCK (Ciona) a tunicate - assembled from EST contigs.

Figure 24. This cartoon illustrates the relative orientation of the CVMtCK Trp264 (red) and the equivalent amino acid (Tyr263, light blue) from the dimeric Tethya (ThMtCK) (unpublished homology model from S. Sona in our lab). The Trp264 is at the core of the interdimer interface in the CVMtCK model, and our site-directed mutagenesis has clearly implicated this amino acid in octamer formation. In Tethya this residue is a Tyr and ThMtCK does not form octamers (Sona et al., 2004). Two monomers of a single dimer (A, a) are shown in pink and the N-terminal of an adjacent dimer (B) is shown in blue. This image was composed by overlaying the relevant residues from both model structures and serves to demonstrate the high level of structural homology that exists between these proteins. This can be seen by examining the "A" helix and the "a" N-term sidechains which are colored pink in Chaetopterus and orange in Tethya and in almost all instances exhibit zero or slight changes in rotomer conformation. Inset indicates the location of these residues and secondary structures within the octamer. This figure was constructed in SPDBV and illustrated in PyMol.

59 relative position of the CV W264 residue (which comparision shows is very similar to the orientation in the SarMtCK PDB file; data not shown) as compared to the equivalent Y264 in the sponge Tethya isoform (from a homology model generated by S. Sona that has not yet been published). This W264 residue (and the equivalent Y264 in Tethya) is coordinated by a helix (242 - 263 in SarMtCK) and is in close apposition to the helix previously mentioned above in our discussion of the salt bridges acting to stabilize the octamer (143 - 158 in SarMtCK). In order to more fully evaluate the role this residue plays in the octamerization of mitochondrial CK, we mutated the Trp264 on CVMtCK to Cys, Tyr, Leu, and a more conservative Phe. DLS analysis of the CV - wild type yielded an Rh of 8.02 nm (+/- 0.054, n=3) and 8.20 nm (+/- 0.079, n=3) for the recombinant SarMtCK - values we demonstrated earlier to correspond to octameric CK. Rabbit muscle MMCK and Tethya MtCK (recombinant Tethya MtCK was kindly provided by S. Sona), both known to exist as dimers, yielded Rh values of 4.06 nm (+/- 0.054, n=3) and 3.96 nm (+/- 0.058, n=3) values we have shown to correspond to a solution containing dimeric CK. Our DLS measurements (as well as FPLC chromatography), indicated that all 4 of the CVMtCK mutants described above destabilized the octamer. In fact, we were unable to detect any degree of octamerization at all, with Rh values ranging from 3.98 nm (+/- 0.505) to 4.27 nm (+/- 0.460) for the mutants (Table 7). Polydispersity (%PD) values, a measure of the homogeneity of the sample in the DLS, indicated that all our samples contained a single population of molecules. Interestingly, the calculated specific activities (µmol·mgATP· min-1·mg-1 Bradford protein) for both the wild type and all four mutants of CVMtCK were not substantially different (Table 7).

Table 7. Hydrodynamic radius (Rh), polydispersity (%PD), and specific activity (S.A.; µmol MgATP · min-1 · mg-1 protein) of various mutations of W264 in recombinant CVMtCK. The data shown here illustrate the role of Trp264 in the maintenance of oligomeric form in CVMtCK. Recombinant SarMtCK and commercially available rabbit MMCK (Roche Applied Science, Indianapolis, IN) provide octameric and dimeric benchmarks, respectively. Recombinant Tethya aurantia (a poriferan, provided by S. Sona in our group) MtCK is the only MtCK isoform reported to date that exists as a dimer under physiologically relevant conditions. Interestingly, in ThMtCK, the residue corresponding to Trp264 is a tyrosine (Sona et al., 2004). (ND = not determined)

Rh (nm) %PD S.A.

CVMtCK W/T 8.02 ± 0.054 20.33 ± 4.9 36.2 SarMtCK W/T 8.20 ± 0.079 17.99 ± 6.0 70.2 Rabbit MMCK 4.06 ± 0.054 15.00 ± 0.9 ND CVMtCK W264C 4.21 ± 0.256 21.21 ± 4.0 29.6 CVMtCK W264F 3.98 ± 0.505 29.53 ± 5.1 26.0 CVMtCK W264L 4.11 ± 0.411 28.89 ± 3.2 32.0 CVMtCK W264 4.27 ± 0.460 19.95 ± 2.2 36.4 ThMtCK W/T 3.96 ± 0.058 10.1 ± 1.9 31.9

61 DISCUSSION

Mitochondrial creatine kinase evolved at a very early stage in the development of metazoans (Suzuki et al., 2004) and it exists primarily as a homo-octamer in all but the most primitive species (Sona et al., 2004). In fact, convincing evidence indicates that the octameric form appeared before the divergence of the deuterostomes and protostomes (Ellington et al., 1998). This, in combination with the high level of conservation across all the known MtCKs identified to date, provides compelling evidence that the octameric state of MtCK may be essential for its physiological role in the cell. It has been suggested that this essential role involves the maintenance of energy homeostasis in one, or both, of two ways – (a) via the unique capacity of MtCK octamers to bind to both mitochondrial membranes in the vicinity of ANT and porin, thereby creating a structural and functional microcompartment favoring creatine phosphorylation which acts to support the vectorial transport of PCr into the cytoplasm, and / or (b) via the hypothesized role in regulation of oxidative phosphorylation by continuously regenerating ADP from ATP and Cr as ATP is produced and transported into the intermembrane space (Biermans et al., 1990; Wallimann et al., 1992; Wyss et al., 1992). Octamers of MtCK have also been implicated in protecting mitochondria from the formation of the permeability transition pore, thereby acting to prevent activation of mitochondrial dependent apoptosis (Dolder et al., 2003). Recently, reactive oxygen species (ROS) have been implicated as possible mediators of mitochondrial dysfunction (Aon et al., 2003; Cortassa et al., 2004); these ROS are known to disassociate and inhibit octameric CK (Cortassa et al., 2004) potentially leading to pathophysiological changes. The observations discussed above suggest that both the presence and the stability of octameric mitochondrial CK within cells have an important place in our understanding of energy budgeting and metabolic homeostasis in metazoan evolution and in metabolism. In the present study, we have shown that the octameric MtCK from the polychaete Chaetopterus variopedatus (CVMtCK) differs substantially in terms of stability as compared to its vertebrate counterparts, particularly with respect to the impact of conversion to the TSAC on quaternary structure. Prior work has shown that avian and mammalian Sar- and UbiMtCKs exist in an

62 equilibrium of octamers and dimers which typically is displaced towards the octameric state at protein concentrations greater than ~ 0.5 mg/ml. Octamers and dimers are clearly interconvertable. These vertebrate octamers readily dissociate into dimers upon dilution with a half-life on the order of days to weeks. We have demonstrated that recombinant CVMtCK displays the same interconvertibility and concentration - dependence of octamerization and stability as has been reported for Sar- and UbiMtCKs (Kaldis et al., 1994; Schlattner & Wallimann, 2000). Furthermore, we have shown that at low protein concentrations, increased temperature up to around 28 oC shifts the octamer: dimer equilibrium in favor of octamers and increases the absolute rate at which equilibrium is attained. This pattern is virtually identical to that of chicken SarMtCK (Kaldis et al., 1994; Gross & Wallimann, 1995) and likely indicates that there is a strong hydrophobic component to octamerization in CVMtCK as has been reported for Sar- and UbiMtCK. Thus, this stabilization of the octameric state seems to be a fundamental feature of MtCKs. The similarity in protein concentration and temperature dependence for these three MtCKs clearly suggests that the dimer - dimer interactions have been conserved even as these animals have diverged over the last ~ 650 million years (Ellington et al., 1998). This observation is further supported by consideration of the fact that the deduced amino acid sequence of the cDNA for CVMtCK is ~ 65 % identical (and 78 -79 % similar) to the amino acid sequences for Sar- and UbiMtCKs (Pineda & Ellington, 1999). In spite of the broad similarities between CV- and vertebrate MtCKs noted above, we have observed at least two major differences in octamer stability between CVMtCK and chicken SarMtCK / human UbiMtCK (and arguably with other vertebrate Sar- and UbiMtCKs as well). The rate constants for CVMtCK octamer dissociation upon dilution

(kdis) and dimer association (kassoc) upon temperature increases were quite high and, interestingly, of nearly the same magnitude for association and for dissociation. In the case of octamer dissociation, rate constants were two orders of magnitude higher and half lives ~ 30 times shorter than corresponding values for chicken SarMtCK at comparable temperatures. In the absence of substrates, octamers of CVMtCK are much less stable than their vertebrate counterpart MtCKs, at least in dilute solution. Interestingly, Wyss et al. (1995) isolated and characterized MtCK from the spermatozoa of the sea urchin

63 Psammechinus miliaris and this MtCK was shown to exist as highly stable octamers which did not readily dissociate except at extremely high salt concentrations. At this writing the putative cDNA of a mitochondrially-targeted CK from another sea urchin, Strongylocentrotus purpuratus, has been reported by NCBI’s genome annotation project, REFSEQ (Pruitt et al., 2005). It would be worthwhile to clone, express, and analyze this MtCK isoform in order to resolve these seemingly conflicting observations. CK exhibits random order bi-bi kinetics; that is, one substrate (either MgATP or creatine) binds to CK and forms a binary complex to which the second substrate binds, forming the catalytically active ternary complex. Products (MgADP and PCr) are released in this same kind of random order, and the enzyme: substrate and enzyme: product complexes are in rapid equilibrium (Blethen, 1972). Work performed as early as 1976 demonstrated that these binding and release events are accompanied by conformational changes and that these changes are, in fact, rate limiting (Rao et al., 1976). More recent work on CK and its homolog AK has shown that these conformational movements are pronounced (Vendelin et al., 2000; Zhou et al., 2000) What do these substrate-induced changes mean in the context of the TSAC- dependent octamer stability we have described for CVMtCK? CVMtCK appears to be much more resistant to dissociation under TSAC conditions than the vertebrate isoforms, and work reported by the Wallimann group clearly shows that UbiMtCK is somewhat more stable than SarMtCK (Schlattner & Wallimann, 2000). A definitive understanding of this issue may have to wait for the publication of an X-ray crystal structure of the octamer in the TSAC. While X-ray crystal structures for apo- and/or ATP-complexed human UbiMtCK (Eder et al., 2000) and chicken SarMtCK (Fritz-Wolf et al., 1996) octamers have appeared, it has not yet been possible to obtain crystals of Sar- or UbiMtCK as the TSAC, even as a dimer. It has been reported that, at CK concentrations above about 1.0 mg/ml, vertebrate octameric CK is still detectable even in the presence of the TSAC components (Gross & Wallimann, 1995). This suggests that the failure to grow diffraction - quality crystals may simply be the result of a nonhomogeneous mixture of CK oligomers, even at the high concentrations typically used in crystal screening. If this is so, one could reasonably argue that the CVMtCK octamer, with its clear resistance to

64 dissociation under TSAC conditions, is at least a good candidate for a systematic crystallization effort. While the existence of equilibria of octamers and dimers at typical crystallization concentrations may provide a sound explanation for the failure to grow TSAC crystals, it does not address the clear and dramatic differences in octamer stability we see. Whether the enzyme is driven into a new equilibrium of octamers and dimers or has undergone dramatic conformational changes requiring extensive de novo screening of crystallization conditions, substrate binding clearly appears to produce large conformational movements in creatine kinase (Forstner et al., 1998). These conformational changes have been described in detail for an obligate cytoplasmic CK dimer from the electric eel Torpedo (Lahiri et al., 2002). Interestingly, this structure shows asymmetrical binding across the dimer (one monomer is bound to MgADP, the other is in the TSAC) using X-ray crystallography. The greatest insight into the degree, complexity, and specificity of substrate- induced changes occurring as substrates bind to enzyme has come with the release of both a TSAC and a substrate-free structure from a monomeric homolog of creatine kinase, AK (Zhou et al., 1997; Zhou et al., 1998; Zhou et al., 1999; Zhou et al., 2000). Release of the TSAC and an apo - AK crystal structure provides a clear and compelling picture of the open and closed state and emphasizes the specificity and complexity of the conformational changes that must accompany substrate binding in phosphagen kinases. In the present report we have shown by two independent measurements of oligomeric state (FPLC and DLS) that the recombinant CVMtCK octamer is highly resistant to dissociation under the same transition-state conditions that led to rapid and near-complete dissociation of the Sar- and UbiMtCK isoforms. There are several possible mechanisms that could explain these clear and dramatic differences. First, it is possible that transition state substrates interact differently with the active site in each of the different MtCKs, inducing less pronounced conformational changes at the dimer - dimer interface in CVMtCK than in Sar- and UbiMtCKs. It is also possible that the dimer - dimer interface itself is more resistant to changes initiated by substrate binding in the active site (Schlattner & Wallimann, 2000). A third and more interesting possibility is suggested by the asymmetrical TSAC /

65 MgADP dimer mentioned above. These data indicate that there may indeed be a kind of structural communication between the active sites of CK. This idea centers on the existence of “communication pathway(s)” between active sites within a dimer and pathways that may exist between an active site and the residues at the dimer-dimer interface. Work published by Fedosov and Belousova (1989) carry this idea to an interesting extreme. They reported that only 50 % of the active sites in the octameric form of MtCK bind MgADP into an enzyme-MgADP-creatine complex while 100% of the active sites in the dimeric cytoplasmic isoform bind the same substrates. It was their conclusion that only half of the active sites in octameric MtCK can form the TSAC while the dimeric form is capable of 100 % occupancy, and they proposed that the reversible transition from octamer to dimer may by responsive to local substrate concentrations and may therefore have a role in the regulation of high-energy compounds in the cell. These reports, and the more substantial work of Kaldis and Wallimann (1995) in which they

report that the kinetic constants [km (PCR), Km (MgATP), Kd (Cr), and Km (Cr)] are 1.5 - 2.0 times lower for the dimeric than for the octameric MtCK, meaning that dimers have a higher substrate affinity than octamers, could be construed as evidence that there is some threshold value of occupied active sites within an octamer that impacts stability. Below this value, the stability of the octamer is relatively unaffected and above this value, the octamer rapidly dissociates. Kaldis and Wallimann (1995) go on to note that the apparent negative cooperativity of CK could also, “hint that the binding sites are somehow structurally connected, which allows communication between them”. Speculation and thought experiments are important and interesting, but real evidence for a correlation between octamer stability and evolutionary trends requires sequence information and real structural data. A substantial body of work has been developed indicating that Ubi- is more stable then SarMtCK under similar conditions (Gross & Wallimann, 1995; Eder et al., 2000; Schlattner et al., 2000; Schlattner & Wallimann, 2000). As can be seen from the crystal structure data visualized in Figure 22 (and detailed in Table 6), the increased stability of the UbiMtCK isoform would easily be predicted from the ten salt bridges across the dimer – dimer interface as compared to the strictly polar interactions acting to hold the SarMtCK octamer together. These salt bridges act through the N-termini amino acids as well as across and between the large

66 and small domains. In addition, a total of 10 hydrogen bonded or polar interactions stabilize the UbiMtCK octamer and only eight are found the SarMtCK isoform. Yet another indication of the increase in stability for the UbiMtCK isoform can be found when considering the roughly twofold increase in interface area in human Ubi- as compared to chicken SarMtCK [~ 1900 Ǻ and ~ 900 Ǻ, respectively (Eder et al., 2000)]. Nooren and Thornton (2003) have clearly demonstrated an almost linear correlation between this interface area and oligomer stability in structures deposited in the PDB database. In UbiMtCK, it is clear that numerous hydrophobic and/or aromatic residues located at the edge of this interface are acting to shield the central electrostatic interactions (the salt bridges mediated by E148, R151, and D155). Site-directed mutagenesis studies conducted by our group and others has provided compelling evidence that the Trp264 residue in both vertebrate isoforms and in CVMtCK has a central role in the stability of the octamer, but the exact influence of this residue has not been immediately apparent. To date, Sar- and UbiMtCK are the only isoforms that have yielded to crystallization studies, and definitive information about our CVMtCK will have to wait for similar crystallization efforts to succeed. While this means we can address the issue of the comparative stability of the CVMtCK octamer with much less certainty, we are not without tools to shed some light on this interesting topic. To this end, we have devoted a large part of this report to a comparative description of the stability of the CVMtCK isoform which shows that real differences exist, including a dramatic increase in the rate of association and dissociation for CVMtCK in the apo state and an almost complete resistance to dimerization in the TSAC. Eder et al. (2000) pointed out that the major departures in the structures of Ubi- and SarMtCKs can be seen in the N terminal residues. Here, the first nine residues assume a completely different conformation with the Ubi- main chain intertwining with an N-terminus from an adjacent dimer forming extensive contacts, including a salt bridge between R7 and E145, whereas the Sar- isoform N-termini do not form any specific interdimer interactions. It is tempting to speculate that interactions between N-terminal residues in CVMtCK may also act to stabilize the octamer but we are limited in our speculation because of the aforementioned lack of a crystal structure. We can, however,

67 point out that our CVMtCK model indicates that 8 polar interactions are at least possible, as compared to two polar interactions seen in the Sar- structure and five interactions, including an electrostatic bridge, the UbiMtCK isoform. We have already noted the salt bridges centered on R151 in the Ubi- structure that have no counterpart in SarMtCK. Our model, and multiple sequence alignments, clearly indicates that CVMtCK is indeed capable of forming these same stabilizing interactions. All of this suggests that the CVMtCK isoform should be, at the very least, more stable than SarMtCK and possibly less stable than UbiMtCK. This is not the case. Our evidence indicates that in the apo state, CVMtCK dissociates much more rapidly than its vertebrate counterparts; however, it also reassociates much more rapidly. This apparent contra- diction leds us to believe that the interactions across the dimer-dimer interface are much more complex than a simple analysis of sequence and homology models can resolve. On the other hand, CVMtCK is extremely resistant to dissociation when in the TSAC, indicating that, at least in the presence of substrates, this enzyme complex is very stable. It is possible that the conformational changes being transmitted to the dimer - dimer interface and affecting octamer stability in the various isoforms as the active sites of MtCK octamers bind substrates may be very different indeed. One could postulate that, in vertebrates, large conformational changes occur at the interface and act to reduce the contact area between adjacent dimers or act to change the relative position of some of the key residues that have been discussed above. It would follow, in this line of reasoning, that the conformational changes transmitted from the active sites to the dimer- dimer interfaces within the CVMtCK isoform are dramatically attenuated and therefore the CV- isoform would demonstrate a significant resistance to dimerization upon substrate binding. If one accepts that octamers of CK indeed play a structural role within the mitochondrial IMS, then one can imagine that there has been an evolutionary trade-off between CK’s catalytic role in energy management which, in this scenario, would be destabilizing as more active sites are occupied, and CK’s structural role, which in this case would be acting to limit the occupancy of the active sites in order to maintain octamer stability. Following this thought to its logical next step would mean that the evolution of the unstable octamers found in vertebrates has been dominated by energetic

68 requirements and, for the much more stable octamers in the polychaete worms, selection has favored the structural role of MtCK within the mitochondria. Evidence for a very stable octamer under TSAC conditions for a mitochondrial isoform isolated from the sperm of the sea urchin Psammechinus miliaris, reported by Wyss et al. (1995) could indicate that energetic considerations did not predominate until the divergence of the chordates from the echinoderms, well after the split between protostomes and deuterostomes and very late indeed after the appearance of the first metazoans. However, one must reconcile this suggestion of an evolutionary trend with the report by our own group of a dimeric MtCK from a the mitochondria of a marine sponge, the most primitive of extant metazoans (Sona et al., 2004). In summary, it is clear that CVMtCK displays great similarities to vertebrate MtCKs in both protein-dependent and temperature-dependent octamer stability but absolute rates of octamer dissociation upon dilution are dramatically faster and the corresponding half life is much shorter. It is also clear that MtCK from Chaetopterus (and from sea urchin) is much more stable in the presence of the TSAC components then Sar- and UbiMtCKs (and presumably other vertebrate MtCKs as well). These are real differences but do these differences have physiological relevance? It has been suggested that octamer - dimer interconversion does not play a direct and significant role in vivo. Gross and Wallimann (1995) provided an interesting discussion on this in which they noted that dissociation of octameric CK within the mitochondria is unlikely to play a role in metabolic regulation for several reasons. First, they note that no effector has been reported that could produce the rapid dissociation seen in the presence of the non-physiological TSAC, and second, they note that the concentration of CK in the IMS has been estimated to be at least 3.5 mg/ml – a value well above any that has been shown to induce dissociation in vitro. Finally, the warm body temperature of birds and mammals would strongly favor the octameric state in the wild type enzyme. It has also been suggested that the cytoplasm of cells is very crowded, with protein concentrations approaching 200 to 300 mg/ml (Fulton, 1982). Undoubtedly, the restricted compartment constituting the mitochondrial IMS is as crowded, if not more so. Although volume space calculations for the IMS are fraught with uncertainty, it seems highly likely that total protein concentrations are quite high. Thus, differences in octamer dissociation behavior

69 upon dilution observed in vitro in CVMtCK reflect true physio-chemical properties but do not necessarily mean that the octamer is less stable in vivo. All of this points to a fundamental, and as yet unanswered, question: Why is CK oligomeric? A thoughtful review of the evidence and discussion provided herein indicates that there may well have been two competing and important evolutionary imperatives acting on the creatine kinase enzyme family. The first of these selective pressures may have been centered on the energy management role that is fundamental and inherent to all phosphagen kinases – that of a buffer acting to reduce the impact of increasing ADP and decreasing ATP concentrations on the free energy yield of hydrolysis in the cells of tissues that are experiencing spatial and/or temporal mismatches between ATP demand and supply during bursts of activity. This would have created a tendency for high substrate affinity and highly accessible active sites. A second and completing pressure on CK may have come from the structural role now clearly seen, especially in the MtCKs; where CK was driven to form higher order oligomeric states in order to mediate contact between the inner and outer membranes of the mitochondria and functionally connect ATP synthesis to PCr production. As a result of this structural role, CK would have become more and more capable of playing a regulatory role in the management of high energy transport and even ATP synthesis itself as the enzyme formed increasingly more complex and sensitive oligomeric states. This evolutionary competition would have a tendency to drive CK towards complex interactions between active sites and oligomeric state – a condition that may very well sum-up the evidence and discussions we have provided in this report.

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79 BIOGRAPHICAL SKETCH

Gregg G Hoffman

Education

B.S. in Biology University of North Carolina at Wilmington Wilmington, NC (2001)

Graduate program - Department of Biological Science University of North Carolina at Wilmington Wilmington, NC (2000-2001) Graduate advisor: Dr. Tom Shafer

Ph.D. in Biological Science Florida State University Tallahassee, FL (2002-present) Graduate advisor: Dr. W. Ross Ellington

Research Interests

Evolution of Enzymes Structure / function relationships in enzymes

Research Skills

RNA extraction, quantification and RTPCR Differential Display of expressed genes Cloning Protein expression in bacterial systems Protein purification by HPLC/FPLC and low pressure chromatography RNA, DNA and protein electrophoresis Homology modeling Bioinformatics Determination of oligomeric state using HPLC and Differential Light Scattering

Academic and Research Positions

Research Assistant Department of Biological Science

80 University of North Carolina - Wilmington (2001)

Teaching Assistant Department of Biological Science Florida State University (2002-2003)

American Heart Association Predoctoral Fellow Department of Biological Science Florida State University (2003-present)

Academic and Professional Societies

Biophysical Society American Association for the Advancement of Science Society for Integrative and Comparative Biology Sigma Xi

Presentations in National Meetings

Determination of species is central to studies of larval recruitment for decapod species in the estuaries of North Carolina (poster), Sigma Xi, Wilmington, NC 2001

A single-step multiplex PCR identification assay to distinguish megalopae of Callinectes sapidus from Callinectes similis in plankton samples (presentation), The Society for Integrative and Comparative Biology, Los Angeles 2001

Bacterial expression of a functional, octameric mitochondrial creatine kinase from a polychaete (poster), The Society for Integrative and Comparative Biology, Toronto 2003

Studies of octamer stability in a primitive mitochondrial creatine kinase (poster), Biophysical Society, Baltimore 2004

Studies of the structural correlates of mitochondrial creatine kinase octamer formation and stability (poster), ASBMB, San Diego 2005

Lectures/Teaching Experience

Anatomy and Physiology Lab I and II (UNCW, 2000- 2001) Developmental Biology Lab (UNCW, 2001) General Biology Lab (FSU 2002, 2003)

81 Publications

Hoffman, G. G. & Ellington, W.R. (2005) Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase. Biochem Biophys Acta 1751, 184-193