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Primary Sequence Analysis and Folding Behavior of EF Hands in Relation to the Mechanism of Action of Troponin C and Calmodulin

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Primary Sequence Analysis and Folding Behavior of EF Hands in Relation to the Mechanism of Action of Troponin C and Calmodulin View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 160, number 1,2 FEBS 0728 August 19 Review Letter-Hypothesis Primary sequence analysis and folding behavior of EF hands in relation to the mechanism of action of troponin C and calmodulin Jean Garitpy and Robert S. Hodges Medical Research Council of Canada Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Received 18 April 1983 The primary sequence of EF hands encodes for elements of secondary structure which includes the presence of hydrophobic and charged domains in the helical regions of these sites. The hydrophobic and charged surfaces located in the N-terminal region of EF hands offer a potential site of interaction with complimentary surfaces on target proteins. Although the binding of calcium to the EF hands of calmodulin and troponin C may lead to a local exposure of these domains, it is the tertiary structure of these proteins that probably dictates the extent to which these domains are exposed and the selectivity of these proteins for target proteins. Calcium-induced folding Calmodulin Sequence analysis Troponin C 1. INTRODUCTION region) of the loop [3]. These results agree with tl folding pattern of EF hands as observed in tl An EF hand can be described as a linear se- crystal structures of carp parvalbumin [l] ar quence of 30-35 amino acids, where the N- and C- bovine ICBP [4]. In the case of calmodulin ar terminal a-helical regions are flanking a 12-residue troponin C, their ability to bind calcium calcium binding loop [l]. To date, at least 6 associated with their role as modulator protein families of EF hand containing proteins have been Calcium binding to their EF hand sites induc established: parvalbumins, troponin C’s, cal- changes in their secondary and tertiary structur modulins, myosin light chains, intestinal calcium These changes in turn, result in the formation ar binding proteins and the brain specific S-100 pro- exposure of hydrophobic surfaces that are in clo teins. The secondary structure analysis of various proximity or represent binding regions to targ EF hand containing proteins has indicated that the proteins such as troponin I and phosphodiesteras C-terminal a-helical region is initiated in the loop This article examines the possible features of E region and that a @-turn region separates both hands that could describe the mechanism of attic helical segments [2]. The &turn probability is par- of these calcium binding proteins. ticularly high for the first 4 residues (+X, + Y 2. GENERATION OF AN AVERAGE Abbreviations: EF hand, second calcium binding site of EF HAND SEQUENCE carp parvalbumin for which the crystal structure is known; ICBP, intestinal calcium binding protein; W-7, The primary sequence of 30 different EF site N-(6-aminohexyl)-Schloro-1-naphthalene sulfonamide exemplifying the 6 families of EF hand containir Published by Elsevier Science Publishers B. V. 00145793/83/$3.00 0 1983 Federation of European Biochemical Societies Volume 160, number 1,2 FEBS LETTERS August 1983 proteins, were aligned to trace common features to include in our tabulation, the inserted residues pre- all EF sites (fig.1). Note that all sequences listed sent in the S-100 and ICBP sequences. The average represent EF hand sites that bind calcium including amino acid sequence deduced from this sequence 4 sequences containing double amino acid inser- analysis is listed in fig.2A. tion sites [5-lo]. A general sequence pattern emerges from our analysis when one tabulates the 3. COMPOSITION OF THE CALCIUM type and frequency of amino acid at each position BINDING LOOP of the EF hand (table 1). Note that the amino acids were grouped by order of polarity [ 111. We did not The primary sequence of a calcium binding loop (12 residues) reflects more than just a series of ++++Loopttttt ligands and side chains designed to maximize the ttx-Helix++++ ttC-Helix+++ binding of Ca2+. The segment Asp,P,Asx,Gly, x Y Z-Y -x -z Asx,Gly, where P is a positive side chain, re- IAKFKAAFDNF* DAlXXGD*ISVKE LGTVKRHL I RSTnC presents the most conserved sequence of the EF KKKLDAIIBKW Df~*II7f=i% FLVMMVRQ II RSTnC KKELAKCFRIF~ DRNADSY*IDAEE LAEIlRAS III RSTnC hand site and is predicted to be a strong&turn for- DKKIBSLMKDG* tiNN@?*IDFDE FLKNNKGV IV RSTnC PKKLQKRIDKW LhFl33GT*VDFDE FLVNNVRC II BCTnC ming region [ 121. The side chains of aspartic acid KKKL%DLFRNF* DKNAiXY*IDLEE LKINLQAT III RCTnC and/or asparagine residues represent calcium coor- KDDIEBLNKDG* MNNDGQ*IDYDE CLEFHKGV IV BCTnC IAEFKBAFSLF* lHXXGT*ITTKE LGTVHRSL I BBCalm dinating ligands at positions +X, + Y and + Z KABLQWIINEV* DAWGT*IDFfE FLTNHARK II BBCalm (fig.2A). In view of the fact that glutamic acid KKKIRMFRVP* CWXSGY*ISAAE LRNVNTNL III BBCalm DKKVDRbIIRU* NIOGDGQ*V~VYEE FVQNUTAK IV BBCalm residues rarely occur in &turn structures [12] and IAKFKKAFPLF* LMXLGT*ITTKE LGTVMRSL I TCalm KAKLQDNINKW DALUXT*IDFPE PLSLMARK II TCalm are only present in this region of the EF hand in the KKKLIKAFKVF+ LWX.XGL*ZTAAE LRNVMTNL III TCalm case of distorted EF sites (site I of S-lOOa,b and DENDIpIIRCA* DIMSlXSi*INYEE F- IV TCalm ADAVDKVMKKL* DEDux;E*VDFQE WVLVML II S-100a ICBPs), one can conclude that other structural re- QKVVDKVUBTL* DsDGDGE*CDFQE FNAFVANI II S-100b quirements besides the type of calcium ligand must ADDVKKAFAII* DQDI(x;F*IEEDE LKLFLQNP II Cparv DGKTKT?LKAG* DsDGoo<*IGVDE FTALVKA III Cparv be met in order to generate a functional EF hand. TRDVKKVFHIL* Lw(DKsGF*IEEEE LGFILKGF II Rparv VKKTXTLMAG* DKlX%‘f*IGADE FSTLVSSS III Rparv This &turn region is shown in fig.2B as part of a PRTLDDLFQKLS DKNGSf*VSFEE FQVLVKKI II PICBP model EF hand. PSTLDBLFEKLI DKNGLXE*VSFEE FQVLVKKI II BICBP IQKFKRAPTVI* LXJNRffiI*IDKED LRDTFAAN I DTNB The average sequence pattern in this part of the IQLFKKAFNNI* iXJNRlXF*IDKfD LHDNLASN I CGRLC EF hand also shows the presence of conserved IQENKKAFSNI* DVDRB3FWSKDD IKAISKQL I SRLC glycine residues which probably play an important Double insertion sites role in the construction of the P-turn segment NRTLINVFHANS CXEGDKYKLSKKE LKKLLQTE I S-loos (position 4) and represent an essential residue WALIDVFNQYS GREGDKtfKLKKSE LKKLINNK I S- 100b PABLKSIFEiiTA AKEGDPtQLSKEE LKQLIQAK I PICBP (position 6) that allows the peptide backbone to PKELKGIFKKYA AKEGDPMJLSKEE LKLLLQTE I BICBP undergo a large change in direction in this part of the EF loop [1,13] and permits the proper folding Fig.1. Primary sequence of 30 EF hands that bind of ligands around the metal [ 141. Noticeable excep- calcium. Abbreviations: RSTnC, rabbit skeletal troponin C [48]; BBCalm, bovine brain calmodulin [49]; tions again include the distorted site I of S-lOOa,b TCahn, Tetruhymena calmodulin [44]; S-1OOa and and ICBPs, and the defunct sites II and III of rab- S-lOOb, (Y- and &subunit chains of bovine brain S-100 bit skeletal muscle alkali light chains [ 15,161. proteins [7,8]; Cparv, carp parvalbumin [SO]; Rparv, Position 8 and to a lesser extent, position 10 of rabbit parvalbumin [5 11; PICBP, porcine intestinal the EF loop region are conserved hydrophobic sites calcium-binding protein [6]; BICBP, bovine intestinal that offer a natural extension to the hydrophobic calcium-binding protein [5]; DTNB, rabbit skeletal region of the C-terminal a-helical region (fig.2C). DTNB light chain [52]; CGRLC, chicken gizzard Their presence in association with the C- and N- regulatory light chain [53]; SRLC, scallop regulatory terminal hydrophobic surfaces may aid toward the light chain [53]; *, insertion site. The italicized portion inner-sphere complex and of the sequence denotes the calcium-binding loop region dehydration of the Ca2+ where X, Y, Z, -Y, -X, -Z represent the calcium suggest a global involvement of the EF site in a coordinating positions of the loop. The roman numerals metal dehydration process. This explanation ap- indicate which of the native protein EF hand sites are pears more plausible than the dehydration mech- listed. anism proposed in [17] which only involves the Volume 160, number 1,2 FEBS LETTERS August 1983 A +---Calcium binding loop-+ -R-terminal region- -C-ten&al region- 12 3 4 5 6 7 8 910 11 X 2 Y 4 2 6 -Y 8 -X10 11 -Z 4 5 6 7 8 9 10 11 ? N N Eli P N BH 8H P BH BH Asp P Asx Gly Asx Gly ? BH N BH N N BH BH ? BH BH ? Ni BH N N N Ni P P _____---c_3---_-~___ --_-- ___~-__________c____-----~ Fig.2. Primary sequence and folding pattern of EF hands. (A) Average sequence of an EF hand. The dotted lines denote regions of pseudo sequence homology. The coordinating residues in the sequence occupy positions X, Y, 2, -Y, -X and -Z. (B) Representation of a model EF hand along its a-carbon backbone (adapted from [lg]). (C) Location of hydrophobic and charged domains in the helical regions of EF hands: (0) residues located in the calcium binding loop; (of residues present in the helical regions. Abbreviutions: BH, bulky hydrophobic residue; N, negatively charged residue; P, positively charged residue; Ni, non-ionic residue; ?, weakly conserved residue; Asp, aspartic acid; Asx, aspartic acid or asparagine; Gly, glycine. residues at positions 7 (least conserved site of the internal sequence homology between the N- and C- loop) and 10 of the EF loop. terminal cu-helical regions (fig.2A; dotted line The omnipresence of a glutamic acid at position regions). Kretsinger [ 13,181 pointed out the - 2 (table 1) may well be finked to its frequent oc- presence of regularly spaced hydrophobes in the currence in cw-helices (Pa, 1.53; strong a-helix helical regions of EF hands.
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