Metal ion binding by proteins

Metal ion binding by proteins Wendy A. Findlay, Gary S. Shaw and Brian D. Sykes University of Alberta, E d m o n t o n , Alberta, C a n a d a The structural importance of metal ion binding to proteins has recently been studied using site-specific mutagenesis and synthetic peptide techniques. Metal binding to synthetic peptides with sequences corresponding to helix-loop-helix calcium-binding sites and 'zinc fingers' induces unstructured peptides to fold into structures similar to domains observed in native proteins. Current Opinion in Structural Biology 1992, 2:57-60

Introduction

Troponin C

Metal ions have many different roles in proteins; as part of the active site of an enzyme, complexed to a substrate, or integral to the structure of the protein. In our opinion, the aspect that has most 'come of age' in the past year is our understanding of the role that metal ions play in determining and stabilizing the structure of proteins, and the concomitant utilization of metal ion binding in protein engineering. In this review, we focus on two metalion-containing structural motifs: helix-loop-helix calciumbinding sites and zinc fingers. In the papers reviewed, we find that synthetic peptides are playing an important role in this field and that metal ion binding alone can fold an unstructured peptide into a structure similar to that of a native protein. These papers also reflect the ever increasing use of NMR to follow structural transitions in proteins and peptides.

Calcium-binding domains For calcium-binding proteins, a common structural motif consists of a loop of 12 amino acids flanked by two a-helices [1o,2], although binding sites consisting of noncontiguous residues also occur. Usually, 2-8 copies of the helix-loop-heM motif are arranged in pairs. In the metal-binding loop, residues 1, 3, 5, 9 and 12 ligate calcium through side-chain carboxyt or amide carbonyl oxygens, and residue 7 through the backbone carbonyl oxygen. The invariant residue Glu12 provides bidentate coordination, yielding a pentagonal bipyramid arrangement about the calcium ion. Recent work on helix-loophelix calcium-binding proteins and synthetic peptides has focused on the contribution of ligating and non-ligating residues to binding affinity, and the role of hydrophobic and electrostatic effects in the structural and functional interaction between calcium-binding sites.

Skeletal troponin C has two domains, each comprising a pair of helix-loop-helix calcium-binding sites. Sites I and II in the amino-terminal domain have a 50-fold lower affinity for calcium than sites M and IV in the carboxyterminal domain. In an attempt to understand this difference, Shaw et a t [3o] synthesized three 34-residue peptides: one with the complete sequence of site M, one in which the ligating residues of site II replaced those of site M, and one in which the whole 12-residue calciumbinding loop of site II replaced that of site M. Replacing the ligating residues alone had little effect on the affinity but replacing the whole loop decreased the affinity by ,-, lO00-fold, suggesting that the non-ligating loop residues may play a major role in determining calcium affinities. The 34-residue synthetic peptide corresponding to site M of troponic C is unfolded in the absence of metal and binds calcium with an affinity of '-'3 IxM [4--]. Calcium binding induces formation~of the helix-loop-helix structure and the peptide d i m e ~ e s to form a pair of calciumbinding sites [5]. The most interesting observation is that binding of a single calcium ion to one synthetic (site M) peptide induces two such peptides to fold [4-]. A 39-residue proteolytic fragment comprising site IV of skeletal troponin C also folds and dimerizes in the presence of calcium. The solution structure of the dimer has been determined by NMR spectroscopy [6°°]. Both the site-M and site-iv dimers have a three-dimensional structure that is very similar to that of the carboxy-terminal domain in the crystal structure of troponin C, including the presence of a short stretch of antiparalle113-sheet between the two Ca2 + -binding peptides and hydrophobic interactions between their helical regions [2].

Calbindin DgK Calbindin D9K has two high-affinity calcium-binding sites - - a normal helix-loop-helix motif (site Il) and a mod-

Abbreviation GRE--glucocorticoid response element.

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Foldingand binding ified one with two extra amino acids in the loop (site I). In order to determine the contribution of electrostatic forces involving non-ligating residues to calcium binding, Ianse et aL [7°] mutated four surface carboxylate groups of calbindin D9K (three in site I and one in site II) to the corresponding amides, so altering the charge on the protein. They compared 1H-NMR assignments of the backbone NH and Call resonances to show that the mutations introduced minor structural changes. Neutralization of three carboxylates in site I reduced the alfinity of the protein for calcium 2000-fold at low ionic strength and 25-fold near physiological ionic strength. For all of the mutants, the cooperativity of calcium binding to the two sites was significantly decreased.

Calmodulin Calmodulin has a similar structure to troponin C, with two domains each comprising two calcium-binding sites. Beckingham [8"] mutated the Glul2 of each calciumbinding loop to a glutanaine, which should remove this residue's capacity for bidentate coordination of the calcium ion with little effect on the overall structure. Using 9-anthroyl choline as a hydrophobic reporter molecule, two sequential conformational changes were observed by fluorescence as being induced by calcium binding to the carboxy- and amino-terminal domains, respectively, of wild-type calmodulin. Mutation of either site 111 or IV essentially eliminates the first conformational change, suggesting that binding to both these sites is required for this transition to occur. In contrast, sites I and II appear to bind calcium independently, as each mutation elinainates a component of the second conformational change rather than the entire transition. Mutation of sites I and II also affects the conformational change initiated by calcium binding to sites 111 and IV, suggesting that the two calcium binding domains of calmodulin interact. In contrast, mutation of sites 11I and IV has little effect on the structural change corresponding to calcium binding to sites I and 11in the amino-terminal domain. Linse et al. [9o] have reported that separated domains of calmodulin retain the calcium binding properties that they have in the intact molecule, and that positive cooperativity of calcium binding is observed within each domain.

Zinc-binding domains Zinc can play either a catalytic or a structural role in proteins. In catalytic sites, zinc binding is tridentate, with histidine being the most common ligand and 'activated' water serving as the fourth ligand. Tetradentate zinc-binding sites are primarily structural and the most common arrangement seems to be discrete sets of four ligands (mainly cysteine but sometimes including one or two histidines) tetrahedrally coordinating each zinc atom [10.]. Exceptions to this rule include metallothionein, which contains seven zinc atoms coordinated by 20 cysteine residues in two zinc clusters, and transcription factor Gal4, which contains two zinc atoms coordinated by six cysteine residues. Recent work on zinc-binding proteins

and peptides has focused on the structure of zinc-binding sites, the arrangement of the metal-binding ligands and their contributions to binding affinity. ¢

Zinc fingers One common motif is the 'zinc finger', which has the sequence (Tyr, Phe)-X-Cys-X2,4-Cys-X3-Phe-X5-Leu-X2-HisXB,4-His (where X can be almost any amino acid). These sequences form small domains in which the zinc ion is tetrahedrally coordinated by the two cysteine and two histidine residues, and the conserved hydrophobic residues form a hydrophobic core [10o]. Using a database of 131 zinc-finger sequences, Krizek et al. [11.] designed and synthesized a 26-residue consensus zinc-finger peptide which had a dissociation constant for zinc of ~ 10-12 M, i.e. much higher than that of any naturally occurring sequence in known proteins. NMR studies showed that the peptide was unfolded in the absence of metal but adopted a structure in the presence of zinc that corresponds to that found for other zinc fingers [12] - - a short two-strand antiparallel [3-sheet followed by a helix with the zinc bound between the two elements of secondary structure. Lowering the pH protonated the second histidine ligand, causing it to dissociate from the Zn 2 + atom and causing local loss of structure. Replacing this histidine with a cysteine residue had little effect on the metal ion affinity of the peptide. Clore et al. [13 o] have observed more than one conformation of a synthetic zinc-finger peptide by 1HNMR spectroscopy, and have proposed that the major form ( > 90% at pH 6-7) corresponds to both histidine residues being ligated to zinc whereas in the two minor forms only one histidine is ligated to zinc. In Xfin-31, a 25-residue synthetic zinc-finger DNA-binding domain, the residues comprising the turn between the ffsheet and the helix have slightly higher mobility than the rest of the backbone of the interior region of the protein, and the two residues at the carboxyl terminus (adjacent to the second ligating histidine) have considerable conformational flexibility [14o],

Zinc4inger-like motifs Steroid hormone receptors have somewhat different zincbinding motifs [15"]. The structures of two protein fragments comprising the DNA-binding domains of the glucocorticoid receptor (71 residues) and oestrogen receptor (84 residues) have been determined in solution by NMR spectroscopy [16,17o.]; each consists of two zincfinger-like motifs forming a structural domain. The metal iigands are all cysteine residues with spacing s'unilar to that in the CC/HH zinc fingers described above and with two zinc atoms coordinated tetrahedrally. Structurally, these motifs are quite different from the CC/HH zinc fingers: the [3-sheet is absent and an et-helix, which begins immediately after the third ligating cysteine residue, extends for 11-13 residues so that the zinc-binding site is at the amino terminus of the helix in each finger. The helices in the two motifs are highly amphipathic and cross at fight angles near their midpoints to form an extensive hydrophobic core similar to that observed for the

Metal ion binding by proteinsFindlay, calcium binding proteins described earlier. The receptors (and the corresponding isolated DNA-binding domains) bind cooperatively as dimers to palindromic DNA with 6 bp half-sites. Residues in one zinc-binding motif are involved in recognition of half-site sequence whereas residues in the other motif are involved in discrimination of the distance between the two half-sites. A model has been proposed [16,17..] in which the helix of the first motif is the recognition helix for binding in the major groove of DN& Synthetic individual zinc-finger peptides (CI and CII) from rat glucocorticoid receptor both bind to DNA corresponding to the glucocorticoid response element (GRE) in a zinc-dependent manner [18oo]. The CI peptide was able to discriminate between the GRE and the very similar oestrogen response element (10-fold lower binding affinity), whereas the CII peptide bound to both with almost equal affinities. When the first cysteine ligand in the CI peptide was replaced by alanine, the peptide was still able to bind zinc but DNA binding was drastically reduced. Studies with the whole DNA-binding domain indicated that the 'double finger' has 30-fold higher affinity for GRE DNA than a single finger. The CI and CII sequences are identical to the finger regions of the glucocorticoid receptor fragment, for which the three-dimensional structure has been determined [16], showing a helix-helix interaction between the fingers. This interaction may stabilize the receptor-DNA complex and yield a higher affinity for the double finger compared with the single finger.

Shaw and Sykes

homologous protein B a c i l l i s s u b t i l i s neutral protease, in which the corresponding loop is shorter and does not bind calcium. The mutant neutral protease was observed to bind three calcium ions, the wild type only two. In the presence of 0.1 M CaCI2, the rate of thermal denaturation of the mutant was observed to be about half that of the wild type at 62 °C. The mutated protein had similar catalytic properties but was much less stable than the wild type at low calcium concentration (for the added site, Kd m l 0 - 4 M ) . These results show that it is feasible to graft a functional loop from one protein into another.

Conclusions The most significant difference between calcium- and zinc-binding sites is that the former can be quite labile; this makes them exquisitely sensitive to variations in calcium concentration, which is appropriate considering the role of calcium binding proteins in the regulation of many physiological processes. In contrast, zinc ions are usually non-labile but can play either a catalytic or structural role in proteins. Both metals can be involved in maintaining the tertiary structure of proteins and can direct the folding of isolated domains or synthetic peptides corresponding to metal-binding sites. In both cases, a key structural feature is the formation of a central hydrophobic core initiated by metal ion binding. We now have a fairly good idea of the structural aspects of these sites; the next step is to understand the thermodynamics and kinetics involved.

Engineering metal-binding sites into proteins One goal of protein engineering is to create new proteins of any desired structure and to incorporate new activities into these structures. The introduction of a metalbinding site could be used to bring together elements of secondary structure in a particular configuration, the formation of which could then potentially be regulated by metal ion binding. Regan and Clarke [19"'] engineered a tetrahedral zincbinding site into a model four-helix bundle protein by introducing a cysteine and a histidine ligand into each of two adjacent helices. The resultant protein was found to bind zinc tightly (dissociation constant, Kd ~ lO-8M), and alkytation of the cysteine sulfhydryts abolished metal binding. Little change in secondary structure accompanied metal binding but the protein became much more stable against denaturation by guanidine hydrochloride. Lieberman and Sasaki [20] used a covalently attached bipyridine to bring together three amphiphilic 15-residue peptides in the presence of Fe(II). Formation of a trisbipyridine metal complex stabilized formation of the three-or-helix bundle. These results show that metal binding can be used to stabilize interactions between elements of secondary structure in a single polypeptide or to bring together structural elements on separate polypeptides. Toma e t aL [21..] have used site-specific mutagenesis to graft a calcium binding loop from thermolysin into the

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest 00 of outstanding interest 1. •

HEIZMANNCW, HUNZIKER W: Intracellular Calcium-binding Proteins: More Sites than Insights. Trends Biochem Sci 1991, 16:98-103.

A good reviewof calcium-b!ndingproteins, which focuses mainlyon 'EF-hand' ones. 2. STRYNADKA NCJ, JAMESMNG: Crystal Structures of the Helixloop-helix Calcium Binding Proteins. Annu Rev Biochem 1989, 58:951-998. 3. SHAWGS, HODGESRS, SYKESBD: Probing the Relationship , Between 0t-Helix Formation and Calcium ~ t y in Troponin C: tH-NMR Studies of Calcium Binding to Synthetic and Variant Site HI Helix-loop-helix Peptides. Biochemistry

1991, 30:8339-8347.

The calcium binding properties of synthetic peptides corresponding to site III of troponin C, and variants incorporating either the ligating residues or the whole calciumbinding loop of site II indicate that nonfigating residues make a major contribution to Ca2+ affinity. 4. SHAw GS, GOLDENLF, HOMES RS, SYKESBD: interactions .0 between Paired Calcium-binding Sites in Proteins: NMR Determination of the Stoichiometry of Calcium Binding to a Synthetic Troponin-C Peptide. J Am t3bem Soc 1991, 113:5557-5563. Binding of a single calcium ion induces folding and association of two syntheticpeptides correspondingto site HIof troponin C to form a two-

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Foldingand binding site homodimer similar in structure to the carboxy-terminal domain of troponin C. 5.

SHAW GS, HODGES RS, SYKES BD: Calcium-induced Peptide Association to Form an Intact Protein Domain: IH NMR Structural Evidence. Science 1990, 249:280-283.

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KAY LE, FORMAN-KAYJO, MCCUBBIN WI), KAY CM: Solution Structure of a Polypeptide Dimer Comprising t h e Fourth Ca 2 + -binding Site o f Troponin C by Nuclear Magnetic Resonance Spectroscopy. BRxhemistry 1991, 30:4323-4333. The structure of a proteolytic fragment corresponding to site 1V of troponin C is determined in the presence of calcium by NMR and found to be a dimer with a structure very similar to that of the carboxy.terminal domain of troponin C. 7.

LINSE S, JOHANSSON C, BRODIN P, GRUNDSTROMT, DRAKENBERG • T, FORSEN S: Electrostatic Contributions to the Binding of Ca 2+ in Calbindin D9K. Biochemistry 1991, 30:154-162. The calcium affinities of eight mutants of calbindin D9K, with one to three surface charges eliminated, are measured at various KC1 concentrations in order to study the role of both protein surface charge and surrounding electrolyte concentration on calcium binding. 8.

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PALMER AG, RANCE M, WRIGHT PE: lntramolecular MoLions of a Zinc Finger DNA-binding Domain from Xfm Characterized by Proton-detected Natural A b u n d a n c e 13C Heteronuclear I ~ Spectroscopy. J Am Chem Soc 1991, 13:4371-4380. Relaxation parameters are obtained for the backbone and side-chain methine carbons of a 25-residue zinc-finger DNA-binding domain complexed with zinc using proton-detected heteronuclear NMR spectroscopy. 15.

SCHWABEJWR, RHODES D: Beyond Zinc Fingers: Steroid Hor-

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m o n e Receptors Have a Novel Structural Motif for DNA Recognition. Trends Biochem Sci 1991, 16:291-296. A good review of the zinc-finger-like motifs in the DNA-binding domain of steroid hormone receptors. 16.

HARDT, KELLENBACHE, BOELENS R, MALER BA, DAH~AN K, FREEDMAN LP, CARLSTEDT-DUKEJ, YAMAMOTOKR, GUSTAFSSON J-A, KAPTEIN R: Solution Structure of the Glucocorticoid Receptor DNA-binding Domain. Science 1990, 249:157-160.

BECKINGHAMK: Use o f Site-directed Mutations in t h e In-

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dividual Ca 2+-bInding Sites of Calmodulin to Examine Ca2+-induced Conformational Changes. J Biol Chem 1991, 266:6027~030. Calmodulin has two domains each containing two Ca 2 + -binding sites. Four single mutants of calmodulin were made with the conserved glutamic acid replaced by glutamine in each binding site. The effect on the two conformational changes induced by calcium binding to calmodulin is monitored in order to study the interactions between the sites. LINSE S, HELMERSSON ,at, FORSEN S: Calcium Binding to Calmodulin and its Globular Domains. J Biol Chem 1991, 266:8050~8054. Rate constants for calcium binding to individual sites in whole calmodulin and in tryptic fragments corresponding to isolated domains are identical, and positive cooperativity is observed between each pair of sites. 9.

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VALLEEBL, AULDDS: Zinc Coordination, Function, and Structure of Zinc Enzymes and O t h e r Proteins. Biochemistry 1990, 29:5647-5659. A good review of zinc-binding proteins. KmZEKBA, AMANN BT, K1LFOILVJ, /VIERKLE DL,, BERG JM: A C o n s e n s u s Zinc Finger Peptide: Design, High-affinity Metal Binding, a p H - d e p e n d e n t Structure, and a His to Cys Seq u e n c e Variant. J Am ~ Soc 1991, 113:4518-4523. In a synthetic consensus peptide based on 131 known zinc-finger sequences, protonation of the second histidine ligand causes the protein to dissociate from the zinc with only local loss of structure. This histidine can be replaced with a cysteine with little effect on metaLbinding affinity. 11. •

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major form ( > 90%), both histidines are ligated to the zinc ion, whereas in the two minor forms only one histidine is ligated.

BERGJM: ZInc Fingers and O t h e r Metal-binding Domains. J Biol Chem 1990, 265:6513-6516.

CLOREGM, OMICHINSKIJG, GRONENBORNAM: Slow Conformational Dynamics at t h e Metal Coordination Site of a Zinc Finger. J A m ~ Soc 1991, 113:4350-4351. Three sets of resonances corresponding to the two ligating histidines are observed using two-dimensional NMIL It is suggested that in the

SCHWABEJWR, NEUHAUSD, RHODES D: Solution Structure of the DNA-binding Domain of t h e • e s t r o g e n Receptor. Nature 1990, 348:458-461. The three-dimensional structure of the 84-residue, DNA-bindmg domain from the •estrogen receptor is determined in solution by twodimensional 1H-NMR techniques. 17. •o

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ARCHERTK, HAGERGL, OMICHINSK~JG: Sequence-specific DNA Binding by Glucocorticoid Receptor Zinc Finger Peptides. Proc Natl Acad Sci USA 1990, 87:7560-7564. Individual zinc-finger peptides from rat glucocorticoid receptor bind to DNA in a sequence-specific manner in the presence of zinc, but have 30-fold lower affinity than a two-finger protein fragment. 19. 00

REGAN L, CLARKE ND: A Tetrahedral Zinc(II)-binding Site Introduced into a Designed Protein. Biochemistry 1990, 29:10878-10883. A zinc-binding site engineered into a model four-helix bundle protein exhibits high affinity for zinc, which stabilizes the protein against denaturation by guanidine hydrochloride. 20.

LIEBERMANM, SASAm T: Iron(H) Organizes a Synthetic Peptide into Three-helix Bundles. J Am Chem Soc 1991, 113:1470-1471.

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TOMA S, CAMPAGNOLI S, MARGARIT I, GIANNA R, GRAND] G, BOLOGNESIM, DE FILIPPIS V, FONTANAA: Grafting of a calcium binding Loop of Thermolysin to Bacillus subtilts Neutral Protease. Biochemistry 1991, 30:97-106. A calcium binding surface loop from thermotysin is grafted into a homologous region of B. subtilisneutral protease; the mutant binds one extra calcium ion, which stabilizes the protein against thermal denaturation.

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WA Findlay, GS Shaw and BD Sykes, Department of Biochemistry and MRC Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta TGG 2H7, Canada.