Biophysical Chemistry 100 (2003) 437–452

Isolation and characterization of the prokaryotic homolog HslVU (ClpQY) from Thermotoga maritima and the crystal structure of HslV૾

Hyun Kyu Songa,1ba , Matthias Bochtler , M. Kamran Azim , Claudia Hartmann a , Robert Huberaa , Ravishankar Ramachandran, *

aAbteilung Strukturforschung, Max-Planck-Institut fur¨ Biochemie, Am Klopferspitz 18a, D-82152 Planegg-Martinsried, Germany bMax-Planck-PAN Joint Junior Research Program, International Institute of Molecular Biology and Cell Biology, ul. Ks. J. Trojdena 4, 02-109 Warsaw, Poland

Received 15 March 2002; accepted 15 May 2002

Abstract

Heat-shock locus VU (HslVU) is an ATP-dependent proteolytic system and a prokaryotic homolog of the proteasome. It consists of HslV, the , and HslU, the ATPase and chaperone. We have cloned, sequenced and expressed both protein components from the hyperthermophile Thermotoga maritima. T. maritima HslU hydrolyzes a variety of nucleotides in a temperature-dependent manner, with the optimum lying between 75 and 80 8C. It is also nucleotide-unspecific for activation of HslV against amidolytic and caseinolytic activity. The Escherichia coli and T. maritima HslU proteins mutually stimulate HslV proteins from both sources, suggesting a conserved activation mechanism. The crystal structure of T. maritima HslV was determined and refined to 2.1-A˚ resolution. The structure of the dodecameric is well conserved compared to those from E. coli and Haemophilus influenzae.A comparison of known HslV structures confirms the presence of a cation-, although its exact role in the proteolytic mechanism of HslV remains unclear. Amongst factors responsible for the thermostability of T. maritima HslV, extensive ionic interactionsysalt-bridge networks, which occur specifically in the T. maritima enzyme in comparison to its mesophilic counterparts, seem to play an important role. ᮊ 2002 Elsevier Science B.V. All rights reserved.

Keywords: HslV; HslU; Proteolytic activity; Crystal structure; Thermostability; Proteasomal homolog

Abbreviations: AAA, ATPases associated with a variety of cellular activities; AOR, aldehyde ferredoxin from Pyrococcus furiosus; EcV, E. coli HslV; EcU, E. coli HslU; EcVU, E. coli HslVU; HiV, H. influenzae HslV; HiU, H. influenzae HslU; HiVU, H. influenzae HslVU; MBP-SulA, maltose-binding protein and SulA fusion protein; r.m.s., root-mean square; T. maritima, Thermotoga maritima; TmV, T. maritima HslV; TmU, T. maritima HslU; TmVU, T. maritima HslVU; Z-GGL-AMC, carbobenzoxy–Gly–Gly–Leu–7-amido-4-methylcoumarin. ૾ This paper is dedicated to the memory of Professor John T. Edsall. *Corresponding author. Tel.: q49-89-8578-2678; Fax: q49-89-8578-3516. E-mail address: [email protected] (R. Ramachandran). 1 Present address: Department of Cancer Biology, Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA. 0301-4622/03/$ - see front matter ᮊ 2002 Elsevier Science B.V. All rights reserved. PII: S0301-4622Ž02.00297-1 438 H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452

1. Introduction w17x. This is in stark contrast to HslVs from E. coli and H. influenzae, which have all been shown HslVU (ClpQY) is a prokaryotic ATP-depend- to exhibit an N-terminal threonine-dependent cat- ent protease that has recently attracted a lot of alytic mechanism. This is surprising, as CodW interest as a simple model system for the eukar- possesses the catalytic threonine residue present in yotic 26Sproteasome w1–4x. Its proteolytic com- HslV from all organisms of known sequence. ponent, HslV, is a dimer of hexamers that forms a In the current studies, we report the cloning and central proteolytic chamber with active sites on expression of HslV and HslU from T. maritima the interior walls of the cavity w1x. As individual (to be called TmV and TmU, respectively). Their subunits share significant sequence w5x and struc- biochemical properties were characterized and the tural similarity w1x with proteasomal b-subunits, crystal structure of TmV was solved at high HslV may be regarded as a 20Sproteasomal core resolution. Comparisons of the available crystal particle lacking the anti-chambers and displaying structures of HslV, amongst other things, throw different symmetry. HslV and proteasomal b-sub- light on the probable factors for thermostability of units are members of the Ntn-family of TmV. and have a threonine residue at the N-terminus after the processing of a methionine residue or a 2. Materials and methods propeptide w1,6–8x. The proteolytic activity of HslV is stimulated by HslU, which possesses 2.1. Cloning and expression ATPase and ‘unfoldase’ activity and belongs to the Hsp100 family of ATPases w9x. The structure T. maritima HslV and HslU were cloned using determination of HslU showed that it exists as a standard polymerase chain reaction (PCR) tech- hexamer, in which each subunit consists of three niques. PCR primers were designed based on the domains termed N-terminal (N), intermediate (I), reported T. maritima DNA sequences w34x, viz. and C-terminal (C), respectively w10x. It also TM0521 and TM0522 for hslV and hslU respec- confirmed it as a member of the family of ATPases tively, such that hslV was flanked by NdeI and associated with a variety of cellular activities¯ BamHI restriction sites while hslU was flanked by (¯¯AAA-ATPase). HslU is therefore a useful model NdeI and NcoI sites. The resultant fragments were system for the base part of the proteasomal 19S ligated into pET-12b or pET-20b expression vec- regulatory cap, which contains six components that tors (Novagen) which had been digested with the belong to the AAA-ATPase family w11x. appropriate restriction (New England In spite of abundant structural and biochemical Biolabs). The integrity of the resultant plasmids information about HslVU, many aspects of the was verified by DNA sequencing. The plasmids enzyme mechanism are still a matter of debate were then transformed into BL21(DE3) or w12x. Not only have different docking modes been B834(DE3). Cells were grown at 37 8C. Expres- observed in HslVU crystal structures from different sion of TmU was induced by the addition of 1 sources, but different quaternary structures, singly mM IPTG at OD (600 nm)s0.7. Maximal expres- and doubly capped species, are also known from sion of TmV was observed when 1 mM IPTG was the crystal structure of the E. coli enzyme added after the cell culture reached saturation. The w10,13,14x. Extensive mutagenesis studies on E. cells were centrifuged and kept frozen at y20 8C coli HslVU w15x could not resolve the ambiguity until further use. in quaternary association. Furthermore, a sodium or potassium ion was implicated in modulating the 2.2. Purification proteolytic activity of HslV by virtue of its prox- imity to the in the crystal structure of The same procedure was used to purify both H. influenzae HslV (to be called HiV) w16x. TmV and TmU. The cell pellet was resuspended Recently CodW, an HslV homolog from B. subtilis, in ice-cold lysis buffer A (20 mM Tris–HCl, pH was shown to be an N-terminal 7.5, 100 mM NaCl) and subsequently disrupted H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452 439 by ultrasonication. A tablet of protease inhibitor protein substrates. In the case of the former sub- cocktail (Roche Molecular Biochemicals),1mg strate, 10 mg of HslV and 25 mg of HslU were of lysozyme and 0.5 mg of Dnase A per liter of incubated for various time periods and tempera- culture were added before ultrasonification. The tures in the presence of 0.4% resorufin-labeled crude cell extract was centrifuged and the resultant casein in buffer U. Undigested substrate was pre- supernatant was immersed in a water bath at 80 cipitated using 5% trichloroacetic acid and 8C for 30 min and then placed in an ice bath for removed by centrifugation. The absorbance of 5 min. Denatured E. coli proteins were removed released peptides in the supernatant fractions was by centrifugation and the supernatant was loaded spectroscopically measured at 574 nm. The deg- onto a Hi-Trap Q-Sepharose anion exchange col- radation of FITC-casein was measured using umn (5 ml, Amersham Pharmacia) which was HPLC. Enzyme samples (15 mg of HslV; 37.5 mg previously equilibrated with buffer A containing 1 of HslU) in buffer U were incubated for 45 min mM EDTA. TmV and TmU eluted at approxi- with 2 mM ATP-gSand 1 mM FITC-casein. The mately 250 and 350 mM NaCl, respectively. The reaction was stopped by the addition of calpain final chromatographic step was performed using a inhibitor I (acetyl–Leu–Leu–norleucinal) to a Superose 6 preparation-grade column (Amersham- final concentration of 1 mM. Pharmacia) previously equilibrated with buffer A Earlier reports have shown that SulA has a high containing 1 mM EDTA and 200 mM sodium tendency to aggregate in vivo, as well as in vitro, chloride. The protein was concentrated to approx- and therefore MBP-SulA (maltose binding protein imately 10 mgyml and stored at 4 8C. N-terminal and SulA fusion protein) was used as the substrate sequencing and electron spray ionization mass w22x. The pMal-p2-SulA plasmid and E. coli strain spectroscopic (ESI-MS) analyses were carried out CSH26 were kindly donated by Dr A. Higashitani to check the integrity of the protein samples. (National Institute of Genetics, Japan). MBP-SulA was overexpressed and purified as previously 2.3. Assays described w22x. The reaction mixture (60 ml) contained 4 mg of MBP-SulA, 1 mg of HslV, 2.5 Proteins were quantified by their absorbance at mg of HslU, 0.02% Triton X-100, 1 mM DTT and 280 nm or by the method of Bradford w18x using 3 mM ATP in buffer U. After 5–7 h of incubation bovine serum albumin as a standard. at various temperatures, the reaction was stopped Nucleotide hydrolytic activity was measured as by adding 35 ml of 50 mM Tris–HCl, pH 6.8, 0.1 described earlier w19x. In this method, the amount M DTT, 2% SDS, 0.1% bromophenol blue and of inorganic phosphate formed is spectroscopically 10% glycerol, and its products were analyzed on determined at 660 nm as a complex with malachite 12% (wyv) slab gels containing SDS w23x. green and ammonium molybdate. Peptide hydrolysis was assayed using carbob- 2.4. Crystallography enzoxy–Gly–Gly–Leu–7-amido-4-methylcou- marin (Z-GGL-AMC; Bachem) as a substrate w3x. Crystals of TmV were grown at room tempera- Enzyme samples (1 mg of HslV; 2 mg of HslU) ture by the hanging-drop vapor-diffusion method. were incubated for 20 min in buffer U (50 mM A hanging drop was prepared by mixing equal ) Tris, pH 7.5, 5 mM MgCl2 , 1 mM adenosine 59- volumes of a 9 mgyml protein solution and the O-(3-thiotriphosphate)(ATP-gS) and2mMZ- reservoir solution w27.5% (wyv) MPD, 200 mM GGL-AMC. The reaction was terminated by the magnesium acetate and 100 mM sodium cacody- addition of 1% SDS to a final volume of 1000 ml late, pH 6.5x. and the fluorescence of the resultant products was Diffraction data were collected using a CCD measured to quantitate them. detector at the BW6 beamline, Deutsche Elektro- Resorufin-labeled casein (Roche Molecular nen Synchrotron Center, Hamburg, Germany. The Biochemicals) w20x or fluorescein isothiocyanate data were processed and scaled using DENZO and (FITC)-labeled casein w21x were used as model SCALEPACK w24x. Crystals belong to an orthorhom- 440 H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452

Table 1 Data collection and refinement statistics

Data collection statistics Space group I222 Cell parameters (A˚ ) as77.53, bs93.75, cs146.17 Resolution rangea (A˚ ) 17.0–2.1 (2.14–2.1) Uniqueytotal reflections 31 188y334 480 Redundancy 10.7 Completenessa (%) 99.3 (99.7) a,b ( ) Rmerge % 5.2 (24.9) Refinement and model statistics c ( ) R-factoryRfree % 19.1y22.9 Resolution range (A˚ ) 17.0–2.1 Modeled residues 171=3 Number of water moleculesyNaq ions 198y3 R.M.S.D. bond lengths (A˚ ) 0.005 R.M.S.D. bond angles (8) 1.24 Average B value (A˚ 2) Main-chainyside-chainywateryNaq 26.8y31.5y35.9y22.7 Ramachandran outlier (%) 0 R.M.S.D., root-mean square deviation. a Values in the parenthesis are for the reflections in the highest-resolution shell (2.14–2.1 A˚ ). b s88 y 88 ( ) ( ) Rmerge hiZZIh,iŽ.NMIh Ž.y hiIh,i Ž., where I h,i is the intensity of the ith measurement of reflection h and NI h M is the average value over multiple measurements. c s y R 8ZZZZZZFocF y8 ZZF o, where R freeis calculated for a 10% test set of reflections. bic space group, I222 and have cell parameters of 3. Results and discussion as77.53 A,˚˚bs93.75 A and cs146.17 A. ˚ Molec- ular replacement was used to obtain initial phases 3.1. Cloning and purification of TmV and TmU using AMoRe w25x and the E. coli HslV (PDB code 1NED) model was used for calculations. The TmV and TmU share 63.3 and 54.1% sequence asymmetric unit contains three subunits of TmV. identity to HslV and HslU from E. coli (to be ( y ) The SIGMAA-weighted 2mFocDF and three- called EcV and EcU), respectively. A sequence fold averaged map calculated after one round of comparison of TmV with HslVs from several rigid-body refinement showed unambiguous den- sources and the b-subunit of the 20Sproteasome sity for nearly all side chains. Models were rebuilt from Thermoplasma acidophilum is shown in Fig. using O w26x and refined using CNS w27x. Engh 1a. TmV has a short extension of five amino acids and Huber parameters w28x were used throughout. preceding two threonine residues and a character- Non-crystallographic symmetry restraints were istic Gly residue at the y1 position. We generated imposed during the refinement cycles, but were constructs withywithout this extension (Table 2). released in the final round. Solvent molecules were This putative propeptide was auto-processed, albeit added using ‘Foc–F ’ maps using CNS. PROCHECK inefficiently, in the E. coli expression system in w29x was used to assess the quality of the model. the former case, resulting in active enzyme. While Programs in the CCP4 suite w30x, CHAIN w31x and the unprocessed form exhibits mixed oligomeric GRASP w32x were used to analyze the structure. states, the processed form exhibits a single oligo- The data collection and the refinement statistics meric state (dodecamer). This situation is analo- are summarized in Table 1. The coordinates and gous to the T. acidophilum 20Sproteasome when structure factors have been deposited in the Protein the a-subunits are not processed w33x. TmU was Data Bank under the accession code 1M4Y. purified according to the same procedure as for H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452 441

Fig. 1. (a) Sequence alignment of T. maritima HslV (TmV), E. coli HslV (EcV), H. influenzae (HiV), B. subtilis CodW (BsV) and the propeptide of the b-subunit of the proteasome from T. acidophilum (TaP). The catalytically important N-terminal threonine residue is marked by a black triangle, and the secondary structural elements, 11 b-strands (cyan arrows), four a-helices (orange ) ( ) ( ) cylinders , and a 310 -helix green cylinder , are indicated above the sequence for TmV. b Sequence alignment of T. maritima HslU (TmU), E. coli HslU (EcU), H. influenzae (HiU), and B. subtilis CodX (BsU). N-, I-, and C-domains are marked as blue, green, and purple bars, respectively. The Walker A- and B-motifs are labeled. In the sequences, identical residues are colored in red and homologous residues are colored in yellow. This figure was produced with ALSCRIPT w49x. 442 H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452

Table 2 Molecular characteristics of T. maritima HslVU expressed in E. coli

Constructs Expression Host cell Expression Oligomera Molecular mass Comments vector level Calculated MS TmU pET-20b B834(DE3) High Hexamer 53052.4 53059.8 Minor degradation bands TmV 156ITGGQQ, 165 TPEDR, 224 SGML Intact pET-20b B834(DE3) Medium Mixture 18932.8 ND Mixture: intact (;80%) and (many states) processed (;20%) formb Processed pET-12b BL21(DE3)pLysSLow Dodecamer 18332.0 18332.0 N-terminal sequencing: ;90% TTIL, ;10% MTTIL ND, not determined. a By gel filtration profile. b By SDS-PAGE band intensity.

TmV outlined in Section 2. It exists as a hexamer, were found to be hydrolyzed faster than ATP, as verified by chromatographic methods (Table 2). whereas CTP was hydrolyzed more slowly com- Both proteins have moderately high expression pared to ATP. In addition, deoxynucleotides were levels in E. coli. also hydrolyzed by TmU. Moreover, TmU was also able to stimulate the amidolytic and caseinol- 3.2. Hydrolysis of nucleotides by TmU ytic activity of TmV in the presence of these other nucleotides (Table 3). TmU hydrolyses ATP and other nucleotides at In contrast to bacterial activators such as EcU comparable rates, as summarized in Table 3 (also and CodX, which were biochemically character- Fig. 2), in a temperature dependent manner. At ized as ATPases, proteasome-activating nucleoti- physiological temperature, the ATPase activity is dase (PAN) from an archaeon, Methanococcus observed to be approximately 10% of the maxi- jannaschii, hydrolyses both ATP and CTP w35x.In mum, which lies between 75 and 80 8C and addition, an N-terminal deletion mutant, PAN(D1- compares well with the optimum growth tempera- 73), possesses general nucleotide-hydrolysis activ- ture of T. maritima, viz. 80 8C w34x. GTP and UTP ity w36x, as observed in the case of TmU (Table

Table 3 Nucleotide dependence of T. maritima HslVU

Pi generationa Z-GGL-AMC Casein TmVqATPgSNA – – TmVUqATP qqqq qqqq qqqq TmVU (yATP) NA qqq qqq TmVUqADP – qqq qq TmVUqAMP-PNP – qqq qqqq TmVUqATPgS– qqqq qqqq TmVUqCTP qq qqqq qqqq TmVUqGTP qqqqq qqqq qqqq TmVUqUTP qqqqqq qqqq qqqqq TmVUqdATP q qqq qqq TmVUqdCTP qqqqq qqqq qqqq TmVUqdGTP qqqq qqqq qqq TmVUqdTTP qqqqqq qqqq qqqq The values are relative to the hydrolytic activity of TmVU at 70 8C as 100% (qqqqqq, 150–200%; qqqqq, 110– 150%; qqqq, 80–110%; qqq, 60–80%; qq40–60%; q, 20–40%; –, -20%); NA, not applicable. a Nucleotide hydrolysis activity was measured in absence of HslV. H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452 443

Fig. 2. Temperature dependence of T. maritima HslVU activity; ATPase, caseinolytic and amidolytic activity is indicated by triangles, squares and circles, respectively. All values are relative to the corresponding activity at 80 8C, which was set to 100% (see Section 2 for assay conditions).

3). It is known that the backbone amide and MBP-SulA. In E. coli, SulA is known to be a carbonyl groups of Ile18 are important determi- natural substrate of HslVU w37x. Currently, a hom- nants for the adenine ring in EcU–nucleotide olog for SulA in T. maritima has not yet been complexes, and the region around Ile18 is quite identified. Nevertheless, it was of interest to deter- well conserved among the several HslUs (Fig. 1b). mine whether TmVU could degrade E. coli SulA, The NTPase activity exhibited by TmU is conse- as this would mean that TmU could also specifi- quently intriguing. Ongoing structural studies of cally recognize this substrate and unfold it. TmU and its complexes with various nucleotide The peptide and casein degradation assays were analogs are expected to clarify some of these less performed in the presence of the non-hydrolyzable understood aspects. ATP analog, ATP-gS, as reported earlier w15x, while the MBP-SulA assay requires ATPase activ- 3.3. Stimulation of the proteolytic activity of TmV ity. TmVU was found to hydrolyze the small by TmU chromogenic peptide without any temperature dependence. The degradation of casein, on the To determine whether TmV and TmU form an other hand, was found to be temperature-dependent active proteolytic system in vitro, their various (Fig. 2). We initially pre-incubated TmVU and E. activity values were examined. Towards this end, coli MBP-SulA for 30 min at 37 8C and then we made use of three different substrates, viz. a increased the assay temperature to 70 8Casthe small chromogenic peptide (Z–Gly–Gly–Leu– ATPase activity of TmU is substantial at higher AMC), casein as a model flexible substrate and temperatures, although it is still approximately 444 H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452

Table 4 Cross-activity of HslVU between T. maritima and E. coli

Complex Z-GGL-AMC Casein MBP-SulA formationa 37 8C508C708C378C508C708C308C708C TmU: TmV No qqqq qqqq qqqq qqq qqqqq qqqqq –– TmU: EcV No qqqq qqqq qqq qq q q –– EcU: TmV No qqqq qqqq qqqq qqq qqqqq qqqqq –– EcU: EcV No qqqq qqqq qqqq qqqq qq qq qqqq – The values are relative to TmVU activity at 70 8C as 100% (qqqqq, 110–150%; qqqq, 80–110%; qqq, 60–80%; qq, 40–60%; q, 20–40%; –, -20%). a By gel filtration.

10% of the maximum at physiological tempera- complex at 37 8C, although the CodX-EcV hybrid tures. It was observed, however, that TmVU could could not degrade the substrate w17x. It was also not degrade E. coli MBP-SulA (Table 4).We reported that CodX-EcV and CodW-EcU hybrids suggest that T. maritima HslU does not recognize could actively hydrolyze Z-GGL-AMC and casein. E. coli SulA; but as a cautionary note it must be It was suggested, therefore, that CodX could not pointed out that MBP-SulA has a tendency to recognize E. coli SulA. aggregate under high-temperature assay conditions, We therefore correspondingly examined the although the assay at physiological temperatures activity of the TmV-EcU and EcV-TmU hybrid is fairly well established. complexes against different substrates. We found We had reported earlier w12x that peptides that for peptide and casein substrates both hybrids derived from the carboxy terminus of HslU stim- were as active as native TmVU and EcVU com- ulate the amidolytic and caseinolytic activity of plexes themselves (Table 4). We then examined HslV to levels observed with wild-type HslU itself. the activity of the TmV-EcU and EcV-TmU Those experiments support a mechanism wherein hybrids against E. coli MBP-SulA. It is expected the carboxy termini of HslU bind to pockets of a priori that since EcU recognizes SulA, at least HslV in order to activate it for proteolytic activity the TmV-EcU hybrid should exhibit activity w13x. In order to check for the same feature in against this substrate. Surprisingly, the TmV-EcU TmVU, we synthesized an octapeptide correspond- hybrid (as also EcV-TmU) did not exhibit any ing to the carboxy terminus of TmU. We found activity against MBP-SulA at both 37 and 70 8C. that this peptide could support amidolytic activity Although the results of the amidolytic and cas- like full-length TmU, thereby suggesting that the einolytic activity values agree well with studies on activation mechanism is conserved across different the B. subtilis complex, the MBP-SulA activity of species. This conclusion is in line with the results the TmV-EcU hybrid does not. Lack of activity of of sequence comparisons of the carboxy termini the TmV-EcU hybrid complex against SulA at 70 of HslU from different sources w12x. We also found 8C might be due to reduced activity of EcU andy that an octapeptide corresponding to the carboxy or aggregation of MBP-SulA at high temperature. terminus of E. coli HslU stimulates the amidolytic On the other hand, the low activity of TmV at 37 activity of TmV. 8C could probably be responsible for the lack of degradation of the MBP-SulA substrate at physi- 3.4. Cross-reactivity between the E. coli and T. ological temperature (Table 4). maritima complexes 3.5. Crystal structure of TmV It is known from studies on B. subtilis HslVU (CodWX) that the CodW-EcU hybrid hydrolyzed The crystal structure of TmV was solved using E. coli SulA is nearly as actively as the EcVU EcV as a model in molecular replacement calcu- H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452 445 lations. The model accounts for all 171 residues Recently w16x, it has been reported that two of TmV, three sodium ions and 198 water mole- alternate sets of inter-subunit interactions in the cules. It has been refined to a free R-value of HiV quaternary structure result in ‘quasi-equiva- 22.9% for data in the range 17.0–2.1 A˚ and has lent’ packing within the assembly. We examined good stereochemistry. The main chain is unambig- the TmV structure and observed that inter-subunit uously defined and the electron density for the interactions of TmV subunits are similar to only side chains of the following residues is either poor one of the two alternate sets of interaction or absent: Phe22, Arg35, Arg86, Arg89 and Arg90. observed in HiV; specifically, where Asp52 makes Among 447 non-glycine and non-proline residues, a salt bridge with Arg83 of an adjacent subunit in the numbers of residues lying in the most favored, the hexameric ring. Consequently, the entrance of additionally allowed, generously allowed and dis- TmV exhibits a circular shape with the distance allowed regions in the Ramachandran plot are 410 across three pairs of Ca atoms among the six (91.7%),37(8.3%), 0 and 0, respectively. The subunits being 19.5, 20.0 and 21.9 A,˚ respectively refinement statistics, as well as model quality (Fig. 5). Although the average diameter as meas- parameters are summarized in Table 1. A represen- ured between Ca atoms is nearly the same for EcV, tative omit map is shown in Fig. 3. HiV and TmV, at 19.9, 19.3 and 20.5 A,˚ respec- The protomer consists of the typical fold exhib- tively, the HiV structure is very elliptical, with ited by b-subunits of the proteasome w6x and it values of 13.1, 19.1 and 25.7 A˚ w14x (17.9, 19.7 forms a double-doughnut-shaped dodecamer w1x. and 22.0 A˚ in the case of EcV) due to its The entrance to the proteolytic chamber of TmV asymmetry. Hence, the TmV structure, in compar- is formed by a hydrogen-bonded turn (followed ison with those of EcV and HiV, supports the ) suggestion that there is inherent flexibility in the by a long helix, H2 and a short 310 -helix, G1. Its average diameter as measured between Ca atoms intersubunit interactions of HslV. qq is 20.5 A,˚ a value that is slightly greater than that K yNa ion-binding sites were identified in of the a-ring (17 A˚ ) and significantly smaller than the crystal structures of HiV, and it was further the b-ring (27 A˚ ) of the archaeal proteasome w6x. proposed that these monovalent ions are likely 2q w x In summary, the tertiary and quaternary structures interchangeable with the divalent Mg ion 14 . of TmV are similar to EcV and HiV (Figs. 4 and TmV crystallizes in the presence of 200 mM 5) w1,13x; however, several differences in details magnesium acetate and 100 mM sodium cacody- do exist. late buffer, and the buffer in which the protein was stored contained 200 mM sodium chloride. We therefore carefully looked for a cation-binding 3.6. Comparison of free HslV structures site analogous to that in HiV, and observed that a solvent peak in each TmV subunit exhibited a A superposition of the Ca atoms of free TmV, geometry inconsistent with water and occurred at EcV and HiV, refined at 2.1, 3.8 and 1.9 A,˚ the same position as the proposed KqqyNa bind- respectively, is shown in Fig. 4. The root-mean ing site (near residues 157, 160 and 163) in HiV, square (R.M.S.) deviation between free TmV and and consistent with that expected for an Naq ion EcV is 0.94 A˚ for 171 Ca atom pairs, with residues w38x. This site in TmV also exhibited distorted 10, 38, 39, 83–90, 93–95, 100, 101, 116–121, octahedral coordination formed by three main- 138–141, 163 and the C-terminus of TmV devi- chain carbonyl oxygen atoms and three water ating by )1.0 A˚ at the Ca atom. Between free molecules, as also reported in the HiV structure TmV and HiV, the R.M.S. deviation is 0.93 A˚ for (Fig. 3b). It is not easy to distinguish between 171 Ca atom pairs, with residues 27, 38, 39, 85– Naq and Mg2q ions in the current structure, but 90, 100, 101, 103, 109–111 and 115–118 of TmV geometric analysis supports that it is likely a exhibiting deviations greater than 1 A˚ at the Ca Naq ion. The mean distance between oxygen and atoms. These residues are located mostly on loops Naq atoms is 2.43 A˚ in the Cambridge Structural (Fig. 1a, Fig. 4). Database and 2.57 A˚ in the Protein Data Bank 446 H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452

Fig. 3. (a) Stereo diagram showing the simulated annealed omit map calculated using data in the range 17–2.1 A˚ and contoured at 3s. Omitted residues (five N-terminal residues) are drawn in thick lines and labeled. Carbon, nitrogen and oxygen atoms are colored green, blue and red, respectively. (b) Stereo diagram of the cation-binding site in TmV (green), EcV (red) and HiV (blue) taken from the first HslV chain in the respective coordinates. Naq and the water molecules are indicated by crosses. Metal–protein and metal–water interactions in TmV are marked. Some residues of TmV are labeled for clarity. H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452 447

Fig. 4. Stereo diagram showing the Ca atom superposition of free HslV structures. Green, red and blue lines represent TmV, EcV and HiV, respectively. Every 20th residue of TmV is labeled. w50x. The mean metal-donor distance between to compare them to understand the causes for the Mg2q and the main-chain carbonyl oxygen is 2.07 high thermostability of TmV. The thermal denatur- w x ( ) A˚ 39 . The mean distance between the ion and ation temperatures Tm of TmV and EcV were oxygen atoms in our structure is 2.40 A˚ (ion– determined by circular dichroism studies to be ) carbonyl oxygen, 2.25 A;˚ ion–water oxygen, 2.54 110 and 70 8C, respectively (unpublished results, A˚ ). This value is closer to that expected for a data not shown). We examined several factors that Naq ion than Mg2q in the database, and moreover are thought to be important determinants of ther- is nearly the same as that observed in the Naq - mostability in proteins (for a recent review see containing HiV structure w16x. w40x), and these are summarized in Table 5 and In view of the HiV and TmV structures, we re- discussed in greater detail below. examined the free EcV and its co-crystal structures, An examination of the primary structures of the which were solved to resolutions of 3.8 and 2.8 three enzymes did not reveal any striking differ- A,˚ respectively w1,10x. Even in the latter structure, ence in the amino acid composition. The number it is difficult to unambiguously distinguish between of residues in TmV is smallest at 171 residues, a solvent site and a KqqyNa ion-binding site. compared to 175 and 174 residues for EcV and Nevertheless, a site assigned earlier to a water HiV, respectively (Fig. 1a, Table 5). It has been molecule occurs at the same position as that of the suggested that shorter loops may contribute to the cation in the HiV and TmV structures (Fig. 3b). resistance of proteins towards thermal unfolding, The exact role of the cation in the proteolytic as observed in the case of citrate synthase from mechanism of HslV is currently unclear w16x. Thermoplasma acidophilum w41x. The number of Arg residues or the ratio Argy(ArgqLys) has 3.7. Structural basis for the thermostability of TmV been reported to be higher in some thermostable proteins w42x. The number of Arg residues in TmV, With the availability of crystal structures of EcV and HiV is 12, 10 and 11, while the Argy HslV from three different sources, it is instructive (ArgqLys) ratio is 0.55, 0.50 and 0.55, respec- 448 H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452

Fig. 5. (a) View along the six-fold axis of the TmV dodecamer. (b) Side view of the dodecamer. Arg89 and Arg90, which form part of a positively charged cluster of residues lining the entrance pore of TmV, are indicated. Positively and negatively charged transparent surfaces are shown in blue and red, respectively. Each polypeptide chain is colored differently for clarity. The figure was drawn using GRASP w32x. H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452 449

Table 5 Comparison of thermostability factors

TmV EcV HiV Total number of residues per chaina 171 175 174 Number of Arg residues 12 10 11 wArgy(ArgqLys)x ratio 0.55 0.50 0.55 Number of deamination sites per chaina 333 Total number of H-bonds (monomer) 5720 (449) 5500 (447) 5736 (460) Volume of cavitiesb3(A˚ ) 0 211 155 Number of cavities 0 20 13 Relative surface areac 0.95 1.03 0.98 Optimized packingd 0.53 0.49 0.51 Number of salt bridges (intrayinter)e 140y52 88y16 80y16 Number of salt bridges per residue 0.094 0.050 0.046 a See Fig. 1a for details. b Probe-accessible volume with probe radius 1.4 A˚ using program GRASP w32x. c0.866s ( s ) Relative surface area AocyA A c15.0=N , where N is the total number of protein atoms . d Efficiency of packing is given by the fraction of atoms in a protein with zero accessible surface area. e Ionic interactions between charged side-chains within 4 A.˚ Calculations are for the dodecamer unless stated otherwise. tively (Table 5). The differences amongst them that TmV has a tightly packed core compared to are not particularly significant. The deamidation EcV and HiV. of asparagine residues is known to be a determi- It is expected that minimization of the ratio of nant of heat inactivation of proteins w43x. The surface area of a protein to its enclosed volume formation of the major product of deamidation, should increase protein stability by simultaneously isoaspartate, frequently occurs at Asn–Gly, Asn– reducing unfavorable surface energy and increasing Ser or Asp–Gly sequences when they lie in regions the packing interactions in the interior, although of the polypeptide that are highly flexible w44x. there are ambiguities about the latter as a general TmV, EcV and HiV have three potential sites of factor responsible for thermotolerance. This is deamidation, and these are located in strictly con- supported by the fact that enzymes from hyperth- served regions of the sequence (Fig. 1a). There- ermophiles have a relatively small solvent-exposed fore, isoaspartate formation is not expected to be surface area w45x. The relative surface area is a the major factor for heat inactivation of EcV and good indicator of the extent of surface area mini- HiV. mization. The observed accessible surface area ( ) 2 An examination of the tertiary and quaternary Ao of TmV is 60 721 A˚ and the expected area ( ) 2 ( s structures shows that the total number of hydrogen Accis calculated to be 63 657 A˚ A bonds per protomer, as well as the dodecamer, 15.0=N0.866, where N is the total number of ( ) ) does not follow expected trends Table 5 . Another protein atoms . The ratio AocyA of 0.95 observed important factor implicated for the protein stability in TmV is relatively low when compared to many is the size and number of cavities in the structure. other protein molecules w45x. Another simple indi- We looked for the presence of cavities in the cator for evaluating packing efficiency is the frac- enzymes from the three sources using GRASP w32x tion of atoms in a protein with zero accessible and a probe radius of 1.4 A.˚ We found that there surface area. For example, a hyperthermophilic are no cavities with a significant probe-accessible tungstopterin enzyme, aldehyde ferredoxin oxido- volume in TmV, while the EcV and HiV structures reductase (AOR) from Pyrococcus furiosus exhib- have significant vacant volumes formed by a its a value of ;0.55, which is significantly higher number of small cavities (Table 5). This suggests than average w45x. In TmV this value is 0.53, 450 H.K. Song et al. / Biophysical Chemistry 100 (2003) 437–452 approximately as high as in AOR. Hence, the ber of intra- and inter-subunit ionic interactionsy relative surface area and packing efficiency in salt-bridge networks observed in TmV appears to TmV follow expected trends for thermostable pro- play a major role. teins (Table 5) and are expected to contribute towards the temperature stability of TmV. Acknowledgments Salt bridges are known to be important deter- w x minants of thermostability in proteins 45–47 . The authors would like to thank H. Bartunik For example, in AOR the number of ‘ion-pairs per and G. Bourenkov for help with data collection; residue’ was found to be more than two-fold the S. Korner¨ for mass spectroscopic analysis; P. average for other proteins (;0.085 vs. ;0.040) w x Zwickl for discussion on the NTPase activity; J. 45 . The factor ‘ion-pairs per residue’ was defined Kaiser for gift of T. maritima genomic DNA; M. as the difference between the numbers of attractive Harding, University of Edinburgh, for discussions and repulsive ionic interactions that occur between on geometry of metal ion–protein interactions; and polar atoms of charged side chains within 4 A,˚ ( w x A. Higashitani National Institute of Genetics, divided by the total number of residues 45 . Japan) for the gift of MBP-SulA fusion protein Moreover, the distribution of residues involved in clone. R.R. and K.A are recipients of fellowships ionic interactions in hyperthermophilic enzymes from The Alexander von Humboldt Foundation. exhibit differences compared to those from meso- philic sources. That is, hyperthermophilic enzymes were found to exhibit extensive salt-bridge net- References works or ion pair clusters w48x. There are a total w1x M. Bochtler, L. Ditzel, M. Groll, R. 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