Protein metal-binding sites John A. Tainer, Victoria A. Roberts and Elizabeth D. Getzoff The Scripps Research Institute, La Jolla, California, USA Metal ions have a role in a variety of important functions in proteins including protein folding, assembly, stability, conformational change, and catalysis. The presence or absence of a given metal ion is crucial to the conformation or activity of over one third of all proteins. Recent developments have been made in the understanding and design of metal-binding sites in proteins, an important and rapidly advancing area of protein engineering. Current Opinion in Biotechnology 1992, 3:378-387

Introduction The explosive growth in information on protein metalsite structures, provided in particular b y recent advances in crystallographic a n d NMR methods, has greatly assisted our understanding of the details of their function. This ability to understand and ultimately control metal binding is of e n o r m o u s importance for biotechnology, science and medicine. The emergence of our understanding and control of protein metal-binding sites is advancing so rapidly that a yearly review is necessary. Here w e will focus on n e w results and aspects that were not covered in our previous review [1"], which provided a brief but comprehensive discussion of protein metal sites along with a review of the work published as far as the beginning of 1991. Particular emphasis is given here to the definition of the geometry of protein metal sites b y atomic Structure determination, to the role of the metal ions in catalysis, and to important recent successes in the design of metal-binding sites in proteins.

General structural chemistry of metal-binding sites Metal ions in proteins are b o u n d b y groups of atoms, termed ligands, that donate an electron pair to the bond. Ligands usually have a neutral or negative charge. Metal sites can be characterized b y the type of metal they bind and b y the coordination number, which is simply the n u m b e r of ligating atoms. Side-chain carboxylate groups (Asp and Glu), thiol and thioether groups (Cys and Met), imidazole groups (His), and hydroxyl groups (Ser, Thr, and Tyr) dominate metal coordination in proteins, although mainchain carbonyl oxygens participate in some sites, particularly those of Ca(II). We reviewed the environment and stereochemistry of side-chain and main-chain ligands last year [1"]. The complexing p o w e r of the metal ion depends u p o n its polarizing power, the ratio of

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charge to ionic radius. As the polarizability of the metal ion increases, protein ligands see an effective increase in high-density positive charge, resulting in an increased interaction. Ionic radii decrease from left to right across the periodic table as a result of the effect of increased nuclear charge. Thus, alkali metals interact weakly with ligands, but the alkaline earth cations Ca(II) and Mg(II) interact more strongly. Divalent cations of the first transition series decrease in size, such that Mn(II) > Fe(II) > Co(II) > Ni(II) > Cu(II) > Zn(II), with formation constants for complexes of these 3d divalent cations with nitrogen-donor ligands tending to increase in the same order. The metal site geometry depends u p o n the n u m b e r of ligands and their stereochemical arrangement, with the n u m b e r of potential ligands depending u p o n the relative sizes of the metal ion and ligating atoms. Octahedral complexes may have two different metal radii resulting from arrangements of electrons: high spin with the m a x i m u m n u m b e r of unpaired electrons gives a larger radius than low spin with the paired electrons maximized. The coordination of metal ions generally approaches the largest n u m b e r possible, and the groups containing the ligating atoms are arranged so as to minimize repulsive interactions b e t w e e n them. The most detailed information on the structural chemistry of metal-binding sites in proteins has c o m e from high-resolution crystallographic and NMR structures. The structural biochemistry of protein metal sites has b e e n considered in depth in a recent comprehensive review [2"]. Metal regulatory mechanisms for proteins have also b e e n reviewed, with the emphasis on structural changes u p o n metal binding [3"]. Metal binding to non-native sites has important applications. Probably the most important and c o m m o n use is the formation of heavy atom derivatives in the determination of Xray crystal structures. Other useful applications include m a p p i n g active sites with metal-ion binding to specific side chains, such as Cys, to inactivate an e n z y m e [4"]. Finally, it should b e noted that metal ions can catalyze the oxidative modification of amino acids such as His and Arg, particularly in reactive environments such as solvent-sequestered active sites [5"].

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Protein metal-binding sites Tainer, Fe-binding sites Fe is the most c o m m o n metal ion in proteins of k n o w n structure. Although these were mainly heme proteins at first, a diverse set of non-berne Fe proteins has n o w also b e e n characterized. Recent reviews on Feprotein chemistry emphasize n e w experimental and theoretical developments in the study of n o n - h e m e Fe proteins [6"1 and theoretical studies of Fe-sulfur clusters in proteins [7"]. Non-heme Fe proteins contain high spin Fe(ID a n d / o r Fe(III) ions (with maximally unpaired d electrons). Previously characterized n o n - h e m e Fe ions in proteins have b e e n found singly in octahedral or trigonal bipyramid geometry, in pairs joined by ~-oxo bridges, and in clusters containing one to four Fe ions with tetrahedral geometry ligated primarily by Cys and inorganic sulfur ligands [6"1. Recent results have shed light on the uptake and oxidation of Fe by ferritin, a protein complex of 24 homologous L- and H-chain four-helix bundle proteins assembled into a hollow shell providing an Fe storage cavity. To crystallize h u m a n H-ferritin it was necessary to create an intermolecular metal-binding site that had b e e n found in horse and rat L-ferritin crystals. The resultant structure reveals a novel Fe center with tetrahedral coordination by Glu27, Glu62, His65, and a water ligand [8"]. This may b e the ferroxidase center that is h y p o thesized to catalyze Fe(II) oxidation to Fe(III) before deposition in the cavity. Mutant recombinant myohemerythrin proteins, expressed in Escherichia coli, give visible absorbance spectra characteristic of the ~t-oxo bridged di-Fe complex in the native folded protein [9"]. A brief perspective o n oxygen activation at the di-Fe center of ribonucleotide reductase [10"] summarizes evidence for intermediates in the proposed enzymatic mechanism. SpectroscoiSic studies of ribonucleotide reductase [11"] have detected two novel intermediates in the assembly of the tyrosyl radical diferric cluster cofactor required for nucleotide reduction to deoxynucleotide. A comparison of the crystallographic structures of native and mutant Azotobacter vinelandii ferrodoxins shows large changes in the crystallographic temperature factors (B-factors) at the [4Fe-4S] cluster in the isomorphous mutant (Cys24 --->Ala) and large structural rearrangements near the [4Fe--4S] cluster, but essentially no change in B-factors for the other mutant (Cys20 --+Ala) [12"'1. Recent crystal structures of complexes of the [4Fe--4S] enzyme aconitase with isocitrate and with nitroisocitrate show both the substrate and the inhibitor bind to the unique Fe atom of the cluster via one hydroxyl and one carbonyl oxygen atom; with a water molecule contributing the sixth ligand [13"]. Three His-carboxylate pairs, implicated in catalysis, a p p e a r to be required for proton transfer reactions involving the oxygen ligands of the Fe. The recently cloned Fe-regulated RNA-binding protein that binds the Fe-responsive element controlling ferritin and transferrin receptor expression has sequence and apparent structural homology to aconitase [14.].

Roberts, Getzoff

Surprisingly, structural studies of h e m e protein cytochrome c peroxidase mutants s h o w that Gin is able to substitute for the proximal Fe ligand His175, maintaining ligation to the heine Fe and almost full enzymatic activity [15"']. Mutations in Asp235, which forms a catalytic triad with His175 and the heine Fe (Fig. 1), modulate the reduction potential without producing large structural changes (DB Goodin, DE McRee, personal communication). In cytochrome c peroxidase, Trp51 is in contact with the h e m e in the distal active-site cavity near the open coordination position of the berne. Six mutants with substitutions at Trp51 all show changes in the state of axial h e m e coordination: most result in a change of the ferric high-spin heme Fe from five-coordinate to six-coordinate form and are more readily converted to a low-spin species at neutral p H [16"]. Changes in the rate-limiting electron-transfer step produce greater than normal activity in three of the mutants.

Fig. I. Close up of the catalytic triad of Asp235, His175, and heme Fe ion in cytochrome c peroxidase. Hydrogen bonds (thin lines) are shown between the polar hydrogen of His175 and the carboxylate of Asp235. Trp191, which is the site of the free radical, also forms a hydrogen bond with the carboxylate of Asp235. The Fe ion is the large grey sphere in the heme ring, which is seen edge on in this view. This figure was made using MOLSCRIPT [53].

Cu-binding sites Cu(II) is a d9 metal ion and is therefore subject to ligand-field stabilization effects. In proteins, Cu(II) prefers square planar or distorted octahedral geometry and Cu(I) prefers tetrahedral geometry. A recent, comprehensive review covers the existing X-ray crystal structures of Cu proteins [17"], which are classified according to their spectral properties into type I, type II, type III, and mixed Cu sites. Type I Cu sites, which function in electron-transfer reactions, are present in the blue c o p p e r proteins, such as plastocyanin, and

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Proteinengineering have a Cu ion b o u n d by a distorted tetrahedral arrangement of a Cys, a Met, and two His ligands. Calculations investigating the interaction between the electron-transfer proteins plastocyanin and cytochrome c suggest that the preferred binding site of cytochrome c is distant from the Cu site of plastocyanin [18"]. This is consistent with evidence for a distance of about 12 from the electron-transfer site of plastocyanin to its Cu ion. The catalytic type II Cu sites found in Cu,Zn superoxide dismutase and galactose oxidase do not share similar geometries. The recently solved crystal structure of galactose oxidase reveals a distorted tetrahedral Cu geometry, with a novel thioether b o n d linking Cys228 to Cu ligand Tyr272 in a stacking interaction with Trp290 [19"']. This arrangement apparently stabilizes the free radical species essential for catalysis, suggesting a new mechanism for mediating electron transfer within metalloenzymes without using additional cofactors. The n e w atomic structure of h u m a n Cu,Zn superoxide dismutase has ten independent subunits in the crystallographic asymmetric unit, allowing accurate assessment of error or variability in active site geometry [20"q. The Cu(II) ligands participate in a h y d r o g e n - b o n d network that joins two different loops on the outside of this ~barrel protein. Ascorbate oxidase, laccase, ceruloplasmin, and nitrite reductase each contain a minimum of four Cu ions: one type I, one type II, and two type IIIs [17"]. Despite the newly discovered diversity in protein Cu(II) sites, Adman [17"] points out that Cu-ion binding sites are found in a very limited subset of structural folds, compared with the more varied environments of the Zn-ion binding sites considered below.

Zn-binding sites Among the first row transition metals, Zn is second only to Fe in terms of abundance and importance in biological molecules. Zn does not have biological redox activity; instead, it usually functions as an electrophilic cofactor in catalytic mechanisms or stabilizes peptide or protein structure. In both roles, Zn(II) can appear as a single b o u n d ion or in a cluster with other metal ions. The structural biochemistry of Zn sites is extensively discussed in a recent review [21-]. Several Zn-containing protein structures solved during the past year shed light on the role of Zn in catalytic mechanisms. Comparison of the refined structures of leucine aminopeptidase [22"], both as the native enzyme and w h e n complexed to the inhibitor bestatin, indicates that both Zn ions in the Zn cluster may be involved in the catalytic mechanism by interacting with the oxygen atoms of the tetrahedral transition state. In addition, one of the Zn ions may provide an activated water or hydroxide, although there is no evidence of Zn-bound solvent in the crystal structures. The crystallographic structure of adenosine deaminase complexed with a transition-state analog reveals the presence of a Zn ion coordinated b y a tetrahedral ar-

ray of three His residues, with an Asp residue and an oxygen atom of the transition-state analog sharing the remaining site [23-]. The Zn ion, which was not k n o w n previously to be a cofactor, has a dual role in both activating a water molecule and aligning the Zn-bound Asp that functions as a general base. A metal triplet, consisting of one Mg and two Zn ions, f o u n d in the crystal structure of alkaline phosphatase [24"] shows the three major types of interactions between carboxylate groups and metal ions: carboxylate as an unidentare ligand, as a bidentate ligand, and as a bridging ligand between two metal ions. Both Zn ions appear to be essential for the catalytic mechanism, while the Mg ion appears to have a structural role. These crystal structures provide new insights into the geometry of Zn relative to the substrate and the process b y which it assists catalysis by stabilizing transition state intermediates and by providing activated nucleophiles. Zn also serves to stabilize protein and peptide structure. The ten subunits of h u m a n Cu,Zn superoxide dismutase s h o w a well ordered helix that is oriented to stabilize, and be stabilized by, the Zn ion [20"]. This helix dipole interaction suggests that Zn binding stabilizes the helix, which contains residues that are important for the electrostatic recognition of the superoxide anion substrate. A combination of crystallographic analysis and site-directed mutagenesis of the allosteric enzyme aspartate carbamoyltransferase from E. coli suggests that the Zn domain may be the pathway of communication between the catalytic and the regulatory domains, because only the Zn domain of each regulatory chain contacts the catalytic domain [25"]. The refined crystal structure of Cd,Zn metallothionein [26"] (Fig. 2), a protein believed to be essential for the maintenance of metal ion homeostasis, is in agreement with structures derived by NMR methods. Without metals, this 61-residue protein, which is one third Cys residues, has no defined three-dimensional structure in solution. The rapid exchange of metals between molecules may be facilitated by dimerization in solution. In the crystal, closely associated pairs of molecules related by the twofold axis may mimic the structure of the dimer in solution. Zn has an important role in stabilizing protein domains that interact with DNA and RNA. Three recent crystallographic strudtures of fragments of DNA complexed to Zn-stabilized DNA-binding domains show details of these interactions. The first crystal structure of a TFIIIAlike Zn-finger peptide complexed to DNA [27"'] has revealed that each of the three fingers binds in a similar orientation relative to the DNA and makes similar contacts with three base pairs of the DNA, consistent with Zn-finger binding in the major groove. Each finger contains a single Zn ion coordinated to two Cys and two His ligands. The crystallographic structure [28"] and several NMR structures (referenced in [28"]) of a fragment of the yeast transcriptional activator, GAL4, shows that the DNA-binding domain has a binuclear cluster of two Zn ions ligated by six Cys ligands, where two of the ligands are shared between the two metal ions. A third DNA-binding motif is defined in the structure of

Protein metal-bindingsitesTainer,

Roberts, Getzoff

Fig. 2. Schematic representation of the metallothionein structure. The N-terminal domain (dark Ca backbone) containing two Zn (large lightcolored spheres) and one Cd (large dark-colored sphere) ions is on the left and the C-terminal domain (light C(z backbone) containing four Cd ions is on the right. Cysteine residues involved in the metal clusters are shown extending from the Cc~ backbone with smaller spheres representing the sulfur atoms. This figure was made with the program MOLSCRIPT [53]. Adapted from [26"].

the glucocorticoid receptor DNA-binding domain that contains twO Zn-nucleated substructures, each with four Cys ligands [29"']. These crystallographic studies with b o u n d DNA and the NMR solution structure studies have e n h a n c e d our understanding of the role of Zn ions in protein-nucleic acid interactions.

Ca-, Mg-, and Mn-binding sites Ca(II), Mg(II), a n d Mn(II) ions are all termed 'hard', reflecting their ligand preferences, their relatively low polarizability, and their small ionic radius. These ions prefer to coordinate oxygen ligands, usually in octahedral geometry. Frequently their function in proteins is to bring together different parts of the protein, orienting functional groups for catalysis and binding, or providing functionally important conformational changes. As a result of the Ca gradient across the cell m e m b r a n e (Ca(II) concentration is high outside and low inside ceils), intracellular Ca-binding sites are important in biological signaling. Ca(ID also mediates biological assembly processes and plays an important role in cataly-

sis. N e w methods in NMR and X-ray structure determination are providing critical insights into Ca(II) sites. NMR spectroscopy has n o w b e e n used to follow Caion-induced conformational changes and the molecular basis for cooperativity in the binding of Ca(II) to bovine calbindin [30"q. In addition, a complete NMR structure of porcine calbindin confirms its similarity to the bovine crystal structure and demonstrates the flexibility of the N-terminal residues [31"q. The threedimensional NMR structure of the complex b e t w e e n Ca(II)-bound calmodulin and a 26-residue synthetic peptide, comprising the calmodulin-binding domain of myosin light-chain kinase, reveals the conformational basis for binding. The long central helix of calmodulin is disrupted into two helices that clamp around residues 3-21 of the b o u n d synthetic peptide, which is folded into a helical conformation [32"q. Lectins represent an important class of Ca(II)- and Mn(II)-binding proteins in which metal ions are required for binding carbohydrates. The m o d e of carbohydrate binding in plant lectins apparently matches that seen in the crystal structures of glucose and mannose complexed to concanavalin A, which contains a ligand-coupled Mn(II) and Ca(II) pair. Replacement of

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Protein engineering Ca(II) with the lanthanide Ho(III) allowed the use of new multi-wavelength phasing methods to determine the crystal structure of a mammalian mannose-binding protein [33"']. This lectin is representative of Ca(II)dependent lectins that function in an antibody-independent host defense against pathogens. The Ca(II)binding region bears n o resemblance to the k n o w n Ca(II)-binding EF-hand motif or the metal sites in plant lectins. Three loops and one p-strand contain the seven ligands, consisting of o n e main-chain carbonyl, one water molecule, and five oxygen atoms from amino acid side chains. In site 1, all of the ligating sidechain atoms come from Asp and Glu residues, while the other site has a mixture of three carboxylate and two Ash carbonyl oxygen atoms. The clustering of the carboxylate ligands suggests that protonation of a carboxyl ligand in the low p H environment of the endosome provides a mechanism for loss of Ca(II)-binding and consequent conformational change and ligand release. Cooperative Ca(II) binding is crucial for the formation of the membrane-binding conformation of the g-carboxyglutamic acid domain of Ca-prothrombin fragment 1, which has b e e n solved at 2.2Aresolution b y X-ray crystallography 0134"q. An array of five Ca(II) ions, each separated b y 4 A and joined b y ~t-oxo bridges, orchestrates the folding of the domain, which is disordered in the apo-protein structure. This m o d e of Ca(II)-ion binding is different from that seen in any other protein structure. X-ray crystal structures of the free and inhibited h u m a n secretory phospholipase A 2 show the two Ca(II) sites seen in other extracellular phospholipase A 2 enzymes. The primary Ca site is hepta-coordinated in a pentagonal bipyramid cage of oxygen ligands. The secondary site is a Ca-binding loop" containing the XCys-Gly-X-Gly sequence conserved in all catalytically active phospholipase A 2 enzymes. The primary Ca plays a role in both substrate binding and catalysis. The secondary Ca site apparently stabilizes the oxyanion indirectly [35"]. The newly determined atomic structure of h u m a n 0~-lactalbumin shows a strong similarity to the b a b o o n enzyme structure [36"']. The Ca(II) coordination is distorted pentagonal bipyramidal and only superficially resembles the EF-hand fold associated with Ca-binding domains in most proteins. Although 0~-lactalbumin can bind a variety of other metal ions, including Zn(II), Cu(II), Co(II), and Mn(II), there are no indications of any additional metal-binding sites. Divalent Mg sites play important roles in enzymatic reactions, particularly those involving phosphate. The Mg(II) can either bind to the substrate or it can have a direct role in formation of the enzyme-substrate complex. Mn(II) is involved in the activation of some hydrolases, such as arginase, and s o m e carboxylases, such as the malic enzyme. An important n e w function of Mg(II) is seen in the crystal structure of the ternary c o m p l e x of ribulose-l,5,-biphosphate carboxylase/oxygenase, Mg(II), and CO 2. The activator CO 2 binds to a Lys side chain to form a carbamate. This labile adduct is stabilized b y a Mg(II) ion that binds the carbamate and Asp and Glu side chains. All these

ligands are located on a single loop of the protein that has a ~ / ~ barrel fold [37"]. Analysis of metal binding to arginase b y atomic absorption spectroscopy indicates that the weakly bound Mn(ID is required for activity but the strongly bound Zn(II), which increases the enzyme's thermostability b y 19°C, participates in the formation of the trimer [38"]. Mn(II) also occurs frequently in phosphate metabolism enzymes, w h e r e it plays a role similar to that of Ca(II). In non-redox proteins, Mn(II) has a similar function to Mg(II) and the two ions are frequently interchangeable. In DNA and RNA synthesis, Mn(II) can replace Mg(II), but results in a loss of specificity. The structure of the ribonuclease H domain has b e e n solved both with and without a metal, showing that there are two divalent cation sites in this domain [39"]. Although Mg(II) is probably the usual cation for this enzyme, the structure was solved with Mn(II), emphasizing the similarity of the two metal ions.

Metal-site design Given the importance of metal ions in biological function, the engineering of metal-binding sites is an important goal for protein design. The challenge is to apply existing structural information to create metal sites that provide a useful function. In the past year, substantial progress has been m a d e in creating metal sites to facilitate protein purification, in building n e w metal sites into existing proteins, and in stabilizing and characterizing de novo metal-binding proteins. The n e w structures reported this year have advanced our understanding of the geometries of protein metal sites and increased the likelihood of continued success in their design. Metal sites are being engineered in proteins and p e p tides to simplify protein purification. Building His-X3His sites in helices of iso-l-cytochrome c [40"q, bovine somatotropin, and insulin-like growth factor has m a d e single-step purifications of the engineered proteins possible [41"]. These metal-chelating sites can b e used to separate correctly folded proteins from those that are incorrectly folded, and are also effective in stabilizing folded proteins. The addition of the peptide His-Trp to the amino terminus of a protein creates a region with a high affinity for immobilized transition metals [42]. The engineered peptide or protein can then be purified b y immobilized metal affinity chromatography. Introduction of a metal site can modulate the characteristics of an enzyme. An interesting n e w review focuses u p o n the engineering of metal sites to regulate e n z y m e activity [43"]. The resultant ability to control e n z y m e activity by manipulating metal-ion availability is e x p e c t e d to have m a n y applications. A surface loop in Bacillus subtilis neutral protease was replaced b y site-directed mutagenesis with a corresponding Ca(II)-binding loop of the h o m o l o g o u s protein thermolysin [44-]. The mutant neutral protease binds three Ca ions, instead of the

Protein metal-binding sites Tainer, Roberts, Getzoff 383 two Ca ions b o u n d to the wild-type enzyme. The stability of the mutant protein relative to wild type is dependent on the concentration of the Ca(II) ion; the mutant is less stable at low Ca concentrations and more stable at high Ca concentrations. An important test of our knowledge of metal-binding site geometries is whether w e can successfully convert one type of metal-binding site to another. One His to Cys mutation was done for each of the metal-binding sites of Cu,Zn superoxide dismutase [45"']. The visible spectrum of the Cu-site mutant was similar to the wildtype except for an additional high energy band, as expected for a S to Cu charge-transfer transition. In contrast, addition of Cu to the Zn-site mutant resulted in the appearance of two strong absorption bands at considerably lower energy, similar to a Cu type I site spectrum. This mutant apparently n o w contains both type I and type II Cu-binding sites and is being characterized further. Knowledge derived from crystal structures is being used to develop methodologies for building new metal sites in existing proteins. A metal-site building computer program [46--] takes as a template the backbone positions of metal-binding ligands from one protein and searches other proteins for that template. Side chains are built into appropriate regions and tested for problems with steric hindrance. Testing the results of this program with site-directed mutagenesis [47"'] indicates that a more extensive understanding of the surrounding environment is needed to prevent alternative modes of metal binding. The metal-ion-assisted self-assembly process is a promising tool for building predetermined secondary and tertiary structures into peptides and proteins. The attachment of bipyridine groups to the N-terminus of 15-amino-acid peptides results in spontaneous self-assembly into three helix bundles u p o n addition of Fe(II) [48"], Ni(ID, Co(II), or Ru(II) [49"]. The use of N-terminal pyridyl groups resulted in spontaneous self-assembly of a 60-residue four-helix l~undle upon addition of Ru(ID [50"'] (Fig. 3). Metal-dependent conformational change is observed for a series of peptides that contain appropriately positioned residues with aminodiacetic acid side chains [51"]. Peptides with three intervening residues between the unnatural amino acids showed the largest enhancement of helicity upon metal addition.

Conclusion The long-term prospects for protein metal-binding site design are extremely promising. The new crystallographic and NMR structures of metalloproteins reported here have extended our knowledge of metalsite geometry. The new information on the orientation and interaction of inhibitors with metal ions in proteins is particularly exciting. These interactions may be suggestive of the geometry in the transition state, and thus provide details that will aid the construction of new catalytic metal sites into proteins such as antibodies [52]. Another major advance comes from crystallographic

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N -G LAQKLLEALQKALA- CONH 2 o

Fig. 3. Computer-generated model of the parallel four-helix bundle metalloprotein. The polypeptide sequence is shown on the bottom, in the single letter amino acid code. This figure was made using MOLSCRIPT [53]. Adapted from [50].

structures of DNA-metalloprotein complexes. These structures provide a basis for constructing proteins that may recognize particular nucleic acid sequences. Further analysis of the n e w structural results reviewed here is needed for the design of metal sites with improved affinities, approaching those of naturally occurring metal-binding sites.

Acknowledgements The work from the authors' laboratories was supported by NIH GM 39345 (IA Tainer), NIH GM37684 (£D Getzoff) and ONR N0001491-J-1885 (ED Getzoff and VA Roberts). We thank the Metalloprotein Structure and Design Group at Scripps for their suggestions of relevant references, and David Case, David Goodin, Duncan McRee, Louis Noodleman, Joan Valentine, and Dennis Winge for providing preprints of their papers before publication.

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References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest TAINERJA, ROBERTS VA, GETZOFF ED: M e t a l B i n d i n g Sites i n P r o t e i n s . Cur7" Opin Biotech 1991, 2:582-591. A review covering crystallographic structures of proteins containing metal ions a n d advances in design of metal sites in peptides and proteins in 1990. 1.

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ALEXANDERH, ALEXANDERS, GETZOFF ED, TAINERJA, GEYSEN HM, LERNER RA: A l t e r i n g t h e A n t i g e n i e i t y o f Proteins. Proc Natl A c a d Sci USA 1992, 89:3352-3356. Mutant recombinant myohemerythrin proteins were expressed in E. coli to test the role of individual critical residues in a highly antigenic site within t h e context of the folded protein. The 328 n m peak characteristic of the p-oxo-bridged di-Fe center was maintained in the recombinant m u t a n t proteins. The immunological results demonstrated that buried side chains can alter antigenic recognition.

GLUSKERJP: S t r u c t u r a l Aspects of Metal Liganding to F u n c t i o n a l G r o u p s i n P r o t e i n s . A d v Prot Chem 1991, 42:1-76. This review provides an excellent introduction to general aspects of interactions b e t w e e n metals a n d protein ligands, including a detailed summary o f the geometry of these interactions.

QUE L JR: O x y g e n A c t i v a t i o n at the Diiron Center o f Ribonueleotide Reductase. Science 1991, 253:273-274. This perspective o n ribonucleotide reductase briefly summarizes the diverse metal centers (p.-oxo-bridged di-Fe, a n d p r o p o s e d di-Mn) a n d organic cofactors (tyrosyl radical, Co-containing adenosylcobalamin, and S-adenosyl methionine) u s e d by these e n z y m e s to catalyze the conversion of ribonudeotides to deoxyribonucleotides. Evidence for proposed reactive intermediates in the m e c h a n i s m o f o x y g e n activation is presented.

3.

11.

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W I N G E DR, DAMERON CT, GEORGE GN: T h e Metallothionein Structural Motif in Gene Expression. Adv Inorg Biochem 1992, 10, in press. This review examines the structural basis of metalloregulation and discusses the mechanisms by w h i c h metal ions m a y activate metalloregulatory proteins. PRINCEMA, FRIEDMANB, GRUSKINEA, SCHROCKRD ni, LLOYD RS: Selective Metal B i n d i n g to Cys-78 Within Endonuclease V Causes an Inhibition of Catalytic Activities Without Altering Nontarget and Target DNA B i n d i n g . f Biol Chem 1991, 266:10686-10693. A series of metal ions (Hg(II), Ag(I), Cu(I)) were s h o w n to inactivate both the pyrimidine dimer-specific DNA glycosylase activity and the s u b s e q u e n t nicking activity of endonuclease V. This paper describes t h e u s e of metals to probe residue function and s h o w s that such results m u s t be further tested by mutagenesis or other m e a n s to discriminate b e t w e e n the direct a n d indirect effects of metal binding. 4.

5,

CLIMENTI, LEVlNE RL: Oxidation o f t h e Active Site o f Glutanaine Synthetase: C o n v e r s i o n o f Arginine-344 to 7-Glutanayl Semialdehyde. Arch Biochem Biophys 1991, 289:371-375. Metal-catalyzed oxidative modification of proteins at His a n d Arg is s h o w n to lead to the appearance of carbonyl groups that can be labeled with fluoresceinamine a n d identified from a tryptic digest. 6.

HOWARDJB, REES DC: Perspectives o n N o n - h e i n e I r o n Protein C h e m i s t r y . A d v Prot Chem 1991, 42:199-280. This overview of the major classes of n o n - h e m e Fe proteins places the e m p h a s i s o n those for w h i c h a crystal structure is available. This review includes a survey of protein Fe centers, a discussion of methods for establishing and characterizing them, a n d a s u m m a r y of recent structural a n d functional results for proteins representing the major Fe classes.

10.

BOLLINGERJM JR., EDMONDSON DE, HUYNH BH, FILLEY J, NORTON JR, STUBBE J: M e c h a n i s m s o f A s s e m b l y o f the T y r o s y l R a d i c a l - d i n u c l e a r I r o n Cluster Cofactor o f Rib o n u c l e o t i d e Reductase. Science 1991, 253:292-298. Stopped-flow absorption spectroscopy and rapid freeze-quench electron paramagnetic resonance spectroscopy are u s e d to identify bt-peroxoferric a n d Fe-coupled radical intermediates in the m e c h a n i s m of tyrosine radical formation. The data suggest that, u n like heme-Fe peroxidases and oxygenases, ribonucleotide reductase does not involve high-valent Fe intermediates in radical formation. 12. •.

SOMANJ, IISMAA S, STOUT CD: C r y s t a l l o g r a p h i c A n a l y s i s o f T w o Site-directed Mutants o f A z o t o b a c t e r v i n e l a n d i i Ferredoxin. J Biol Chem 1991, 266:21558-21562. Stlucmres of t h e Cys20 a n d Cys24 to alanine mutants of this 7Fe ferredoxin are compared. Similar conformational changes are observed at residue 24, w h i c h in Cys20 to Ala b e c o m e s a replacement ligand for the [4Fe-4S] cluster. The solvent accessibility of the Fe atoms in the [3Fe-4S] and [4Fe-4S] dusters is similar in the wild-type protein a n d both mutants. 13. •.

LAUBLEH, KENNEDY MC, BEINERT H, STOUT CD: C r y s t a l Structures o f Aconitase w i t h Isocitrate a n d Nitroisocitrate Bound. Biochemistry 1992, 31:2735-2748. T h e 2.1_~ resolution crystal structures of mitochondriai aconitase complexed with isocitrate and nitroisocitrate were solved a n d refined. The u n i q u e Fe of the [4Fe-4S] cluster that binds t h e subs*rate a n d the inhibitor is pulled away from the corner of the cubane. At least 23 residues from all four d o m a i n s of aconitase contribute to the active site, including three His-carboxylate pairs implicated in catalysis.

NOODLEMANL, CASE DA: D e n s i t y - f u n c t i o n a l T h e o r y o f Spin Polarization and Spin Coupling in Iron-sulfur Clusters. Adv Inorg Chem 1992, 38:423-470. This comprehensive review reports recent progress towards the development of a unified theoretical description of the electronic structure and spin interactions of Fe-sulfur clusters in proteins. Advances made both with quantitative density-functional calculations a n d with phenomenological modeling are discussed.

ROUAULTTA, STOUT CD, KAPTAIN S, HARFORDJB, KLAUSNER RD: Structural Relationship b e t w e e n an Iron-regulated R N A - B i n d i n g Protein (IRE-BP) and Aeonitase: F u n c t i o n a l I m p l i c a t i o n s . Cell 1991, 64:881--883. T h e s e q u e n c e o f the h u m a n Fe-responsive element binding protein (IRE-BP) has be~n m a p p e d onto t h e three-dimensional crystallographic structure of the h o m o l o g o u s enzyme aconitase. Active-site residues are conserved, including t h e three Cys ligands o f the Fe-S center. The authors speculate that t h e aconitases and t h e iron-regulated RNA-binding protein are m e m b e r s of a family of proteins with dynamic Fe-S centers that sense alterations in Fe availability.

8. •.

15. •.

7.

LAWSON DM, ARTYMIUK PJ, YEWDALL SJ, SMITH JMA, LMNGSTONEJC, TREFFRY A, LUZZAGO A, LEVI S, AROSIO P, CESARENI G, ET AL.: S o l v i n g the Structure o f H u m a n H F e r r i t i n b y G e n e t i c a l l y Engineering Intermolecular C r y s t a l C o n t a c t s . Nature 1991, 349:541-544. Based on examination of the Cd(II)-bridged intermolecular crystal contacts in horse and rat L-chain ferritins, site-directed mutagenesis of Asp84-->Gln in h u m a n H-chain ferritin introduced a Ca(II)-bridged crystal contact. The subsequently determined crystal structure identified a novel Fe-bJnding site, w h i c h is e m b e d d e d within each 4-helix bundle protein and has additional ligand stabilization by b o u n d water molecules. The authors suggest that this site is associated with ferroxidase activity, accounting for the rapid uptake of Fe.

14.

SU-NDARAMOORTHYM, CHOUDHURY K, EDWARDS SL, POULOS TL: C r y s t a l Structure and P r e l i m i n a r y F u n c t i o n a l A n a l ysis of the Cytochrome c Peroxidase His175Gln Proxim a l Ligand Mutant. J A m Chem Soc 1991, 113:7755-7757. Wild-type a n d mutant recombinant yeast cytochrome c peroxidase containing a Gin replacement for proximal ligand His175 were expressed and purified from E. coll. The mutant retains nearly full activity as estimated from steady-state kca t values. Comparison of the crystallographic structures of the wild-type and m u t a n t proteins, determined at 2 A resolution with a crystallographic residual error (R-factor) of 18%, s h o w e d adjustments of the replacement Gin side chain to maintain both the b o n d with Fe (apparently coordinated by the side-chain oxygen) and the h y d r o g e n b o n d to Asp235.

Protein metal-binding sites Tainer, Roberts, Getzoff 385 16.

GOODIN DB, DAVIDSON MG, ROE JA, MAUK AG, SMITH M: A m i n o Acid Substitutions at Tryptophan-51 o f C y t o c h r o m e c Peroxidase: Effects o n C o o r d i n a t i o n , Species Preference for C y t o c h r o m e c, a n d E l e c t r o n Transfer. Biochemistry 1991, 30:49534962. Amino acid replacements of Trp51, which is in contact with the heme of yeast cytochrome c peroxidase, have significant effects on the kinetics and coordination state of the enzyme. Optical and electron paramagnetic resonance spectroscopy on six mutants at this site, in which Trp51 is replaced by Phe, Met, Thr, Cys, Ala, and Gly, s h o w e d shifts from the five-coordinate to six-coordinate form and slight increases in the asymmetry of the heine ligand field. ADMANET: Copper Protein Structures. Adv Prot Chem 1991, 42:145-197. A comprehensive review of Cu-binding sites in proteins. 17.

18.

ROBERTSVA, FREEMAN HC, OLSON AJ, TAINER JA, GETZOFF ED: Electrostatic Orientation of the Electron-transfer Complex B e t w e e n Plastocyanin and C y t o c h r o m e c. J Biol Chem 1991, 266:13431-13441. Computer graphics and systematic search methods were used to determine the most favorable precollision orientations of the two proteins as determined by electrostatic forces. Docked complexes derived from the precollision orientations showed that productive dockings should occur for two complexes, suggesting that a unique orientation may not be required in some electron-transfer reactions. 19. •.

ITO N, PHILHPS SEV, STEVENS C, OGEL ZB, MCPHERSON MJ, KEEN JN, YADAVKDS, KNOWLESPF: Novel Thioether B o n d Revealed b y a 1.7A Crystal Structure of Galactose Oxidase. Nature 1991, 350:87-90, Galactose oxidase possesses a unique mononuclear Cu site essential for catalyzing a two-electron transfer during the oxidation of primary alcohols to corresponding aldehydes. The authors report the crystal structure of galactose oxidase at 1.7,~ resolution. The Cu site involves a novel thioether b o n d linking Cys228 and Tyr272 in a stacking interaction with Trp290. This feature may represent a n e w mechanism for mediating electron transfer in metalloenzymes without exogenous eofactors. PARGEHE, HALLEWELL RA, TANNERJA: Atomic Structures o f Wild-type and Thermostable Mutant R e c o m b i n a n t H u m a n Cu,Zn Superoxide Dismutase. Proc Natl A c a d Sci USA 1992, 89:61094113. The ten subunits, which occur in several different crystal environments, in the crystal structure of h u m a n Cu,Zn superoxide dismutase were solved independently, providing an accurate characterization of those lea.rares that are important for enzyme stability and function regardless of crystal environment. The environment of the Zn ion is stabilized by conserved hydrogen bonds in the loop containing the Zn ligands and by a helix dipole interaction with the Zn site. A second structure of a two-site mutant shows that a Cys to Set mutation improves side-chain to main-chain hydrogen bonds, which may account for the increased thermostability of this mutant relative to the wild-type. 20. •.

CHRISTIANSONDW: The Structural Biology of Zinc. Adv Prot Chem 1991, 42:281-355. The author presents a comprehensive review of the structural biochemistry of Zn sites in protein structures and the relevance of metalloprotein structures to the engineering of Zn-binding sites. 21.

22.

•.

BURLEYSK, DAVID PR, LIPSCOMB WN: I.eueine Arnltxopeptidase: Bestatin Inhibition and a Model f o r Enzynxecatalyzed Peptide Hydrolysis. Proc Natl Acad Sct USA

1991, 88:691645920. The m o d e of bestatin inhibition of leucine aminopeptidase is discussed and correlated with biochemical studies of bestatin analogues. A novel feature revealed by the refined structure is a Lys side chain tigating to one of the Zn ions in the two-Zn duster. A general base mechanism involving a Zn-activated water or hydroxide is proposed. 23. •,

WILSON DK, RUDOLPH FB, QUIOCHO FA: A t o m i c Struct u r e o f Adenosine Deaminase C o m p l e x e d w i t h a Transition-state Analog: Understanding Catalysis and Imtnunodeficiency Mutations. Science 1991, 252:1278-1284.

Deficiency of adenosine deaminase is associated with severe combined immunodeficiency disease. The authors have solved the structure of this enzyme, revealing that a Zn ion is a cofactor, and they propose a detailed catalytic mechanism based on the active site environment. KIM EE, WYCKOFF HW: Reaction Mechanism of Alkaline Phosphatase Based o n Crystal Structures. J Mol Biol 1991, 218:449464. This refined structure (2.02~ resolution) reveals details about the interesting three-metal cluster of one Mg and two Zn ions and its involvement in catalysis and structure. The crystallographic structure is compared with previous data from NMR spectroscopy and low-resolution crystallographic structures. 24. ..

25.

STEVENS RC, CHOOK YM, CHO CY, LIPSCOMB WN, KANTROWlTZ ER: E s c b e r i c h i a coil Aspartate Carbanloyltransferase: The Probing o f Crystal Structure A n a l y s i s v i a Site-specific Mutagenesis. Prot Eng 1991, 4:391408. This review compiles and analyzes the data from all published sitespecific mutagenesis experiments on aspartate carbamoyltransferase and puts these data in the context of the k n o w n crystal structures. Bound Zn appears to be essential for proper folding. One proposal is that more mutagenesis experiments need to be done in the interface between the Zn domain and the allosteric domains to understand the basis for signal transduction. 26. •.

ROBBINSAN, MCREE DE, WILLIAMSONM, COLLETTSA, XUONG Nit, FUREY WF, WANG BC, STOUT CD: Refined Crystal Structure of Cd,Zn M e t a l l o t h i o n e i n at 2.0A Resolution. J Mol Biol 1991, 221:1269-1293. High-resolution data was essential to solve this interesting structure of a 61-amino-acid peptide consisting of one third Cys residues and seven b o u n d metal ions. Short cysteine peptide sequences repeated in the structure adopt restricted conformations that favor the formation of amide to sulfur hydrogen bonds. Every Cys residue is ligated to a metal ion. PAVLETICHN-P, PABO CO: Zinc Finger-DNA Recognition: Crystal Structure o f a Zif268-DNA Complex at 2.1A. Science 1991, 252:809-817. This is the first reported crystallographic structure of a Zn finger. The TFIIIA-like peptide contains three fingers that bind in the major groove of B-DNA with each in a similar orientation relative to the DNA. The high-resolution structure provides a framework for understanding h o w Zn fingers recognize DNA. 27. •.

28. •.

MARMORSTEINR, CAREYM, PTASHNE M, HARRISON SC: DNA

Recognition b y GAL4: Structure o f a Protein-DNA Complex. Nature 1992, 356:408--414.

This unusual Zn-binding domain contains a unique cluster of two Zn ions b o u n d by six Cys ligands. When b o u n d to DNA, the GAL4 fragment dimerizes for palindromic recognition of DNA with a DNA spacer between the two b o u n d domains that may allow another protein to bind coordinately with GAL4. 29.

LuISIBF, XU WX, OTWlNOWSKI Z, FREEDMANLP, YAMAMOTO KR, SIGLERPB: C r y s t a l l o g r a p h i c A n a l y s i s of the Interaction of the Glucocorticoid Receptor w i t h DNA. Nature 1991, 352:497-505. The structure provides details of the two Zn-nudeated domains of a receptor fragment. One Zn stabilizes the DNA-binding domain while the other stabilizes the dimerization domain. Dimerization for palindromic recognition of DNA occurs u p o n DNA binding. The importance of the length of the DNA spacer is s h o w n by one structure with an extra intervening DNA base pair. •.

30. ..

AKKEM, FORSEN S, CHAZIN WJ: Molecular Basis for Co-

31. •.

AKKEM, DRAKENBERGT, CHAZIN WJ: Three-dimensional Solution Structure of Caa+-loaded Porcine C a l b i n d i n D9K Determined b y Nuclear Magnetic Resonance Spectroscopy. Biochemistry 1992, 31:1011-1020.

o p e r a t i v i t y i n Ca 2+ B i n d i n g t o C a l b i n d i n D9K. J Mol Biol 1991, 220:173-189. This NMR study shows the structural differences between apo-, halfsaturated, and fully saturated species of calbindin using a Cd(II) substitution for the half-saturated variant. Most of the stmctural changes occur u p o n binding of the first metal ion.

386

Protein engineering The NMR results demonstrate that the N-terminal residues missing from the most c o m m o n l y studied form of calbindin are highly flexible so t h e y do not influence the other structural regions. The protein consists primarily of four helices in two pairs of helix-loophelix motifs joined by a linker segment. 32. •.

IKURAM, CLORE GM, GRONENBORN AM, ZHU G, KLEE CB, BAX A: S o l u t i o n S t r u c t u r e o f a C a l t n o d u l i n - t a r g e t Pept i d e C o m p l e x b y M u l t i d i m e n s i o n a l NMR. Science 1992, 256:632--638. The NMR results provide a three-dimensional solution structure of the complex b e t w e e n Ca-bound calmodulin a n d a 26-residue peptide comprising the calmodulin binding d o m a i n of the skeletal muscle tight-chain kinase. The long central helix is disrupted into two helices connected by a long flexible loop that allows the two domains to clamp around the b o u n d peptide. 33. •.

WEIS WI, KAHN R, FOURME R, DRICKAMERK, HENDRICKSON WA: S t r u c t u r e o f t h e Caleiura-dependent L e c t i n Dom a i n f r o m a Rat M a n n o s e - b i n d i n g Protein D e t e r m i n e d b y MAD P h a s i n g . Science 1991, 254:1608-1615. The X-my crystal structure o f the carbohydrate-recognition domain of a rat mannose-binding protein was determined as the Ho-substituted complex by multiwavelength a n o m a l o u s dispersion. The protein's u n u s u a l fold consists of two distinct regions, one of which contains extensive non-regular secondary structure stabilized by the two Ho atoms. This paper demonstrates a general approach to the determination of Ca(II) protein crystal structures a n d discovers a n e w structural motif for Ca(II) binding. SORIANO-GARCIAM, PADMANABHANK, DE VOS AM, TULINSKY A: T h e Ca 2+ I o n a n d M e m b r a n e Binding Structure o f t h e Gla D o m a i n o f C a - p r o t h r o m b i n F r a g m e n t 1. Biochemistry 1992, 31:2554-2566. The X-ray structure of the Ca-prothrombin fragment 1 s h o w s a unique m o d e of Ca(II) binding for 7 Ca(II) ions, including a polymeric array o f five Ca(II) ions separated b y about 4A.

D o m a i n o f HIV-1 Reverse Transcriptase. Science 1991, 252:88-95. T h e crystal structure for the ribonuclease H domain of h u m a n immunodeficiency virus-1 reverse transcriptase, which is crucial in the life cycle of retroviruses, was determined at 2.4 A resolution. T w o divalent cations bind in the active site. Although the structure is similar to the ribonuclease H of Escherichia coli, the isolated ribonuclease It domain of h u m a n immunodeficiency virus-1 is not catalytically active. 40. •.

TODD RJ, VAN DAM ME, CASlMIRO D, Iq.AYMOREBL, ARNOLD FH: C u ( l I ) - b i r t d i n g P r o p e r t i e s o f a C y t o c h r o m e c W i t h a S y n t h e t i c M e t a l - b i n d i n g Site: His-X3-His i n an or-Helix. Proteins 1991, 10:156-161. A metal-binding site consisting of two His residues positioned HisX3-His was built into iso-l-cytochrome c on the surface of a helix. The mutations did n o t affect activity or expression of the protein. The metal affinity of the synthetic protein is sensitive to environmental conditions, s u c h as pH, that alter the flexibility of the helix structure. 41.

ARNOLDFH, HAYMORE BL: E n g i n e e r e d M e t a l - b i n d i n g Prot e i n s : P u r i f i c a t i o n to Protein Folding. Science 1991, 252:1796-1797. T h e authors s u m m a r i z e the metal-binding affinity conferred o n a prorein u p o n the creation of a His-X3-His o n an e x p o s e d cz-hetical segm e n t of a protein. Implications for protein purification a n d protein stabilization are discussed. 42.

34. •.

35. •.

SCOTFDL, WHITE SP, BROWNINGJL, ROSAJJ, GELB i N , SlGLER PB: S t r u c t u r e s o f Free and Inhibited H u m a n S e c r ~ o r y P h o s p h o l i p a s e A 2 f r o m Infl~lrnrnatory E x u d a t e . Science 1991, 254:1007-1010. The 2.1 X crystal structures of recombinant h u m a n secretory phospholipase A2, both in the presence a n d absence of a transitionstate analog, suggest that the chemistry of catalysis is identical to that suggested by crystal structures o f other phospholipases. The shape of the hydrophobic channel of h u m a n secretory phospholipase A2, however, is uniquely modulated by substrate binding. 36. •.

ACHARYAKR, PEN J, STUART DI, PHILLIPS DC, FENNA RE: C r y s t a l S t r u c t u r e o f I t t l m a a 0~-Laetalbumin at 1.7A Resolution. J Mol Biol 1991, 221:571-581. The X-ray structure of h u m a n 0t-lactalbumin with b o u n d Ca(II) shows the striking structural resemblance b e t w e e n tx-lactalbumins a n d C-type lysozymes a n d the implied evolutionary relationship between these protein classes. 37. •.

LUNDQVIST T, SCHNEIDER G: C r y s t a l S t r u c t u r e o f the T e r n a r y C o m p l e x o f Ribulose-l,5-Bisphosp.l.l.l.l~te Carb o x y l a s e , Mg(II), a n d Activator CO 2 at 2.3A Resolution. Biochemistry 1991, 30:904-908. The activated ternary complex, enzyme-CO2-Mg(II) , of the dimeric ribulose-l,5-bisphosphate carboxylase/oxygenase from Rhodospirtllum r u b r u m was compared with the native non-activated enzyme. The hydrogen-bonding pattern in the vicinity of the activator site is completely altered. 38. •.

GREEN SM, GINSBURG A, LEWIS MS, HENSLEY P: Roles o f M e t a l I o n s i n the Maintenance o f t h e Tertiary and Q u a t e r n a r y s t r u c t u r e o f A r g i n a s e f r o m Saccharomyces cerevi$iae. J Biol Chem 1991, 266:21474-21481. This paper uses atomic absorption m e t h o d s to s h o w that the tightly b o u n d Zn(II) provides significant therrnostability by promoting trimer assembly, but the weakly b o u n d Mn(II) is necessary for activity. 39. •.

DAVIESJF II, HOSTOMSKA Z, HOSTOMSKY Z, JORDAN SR, MATrVlEWSDA: C r y s t a l S t r u c t u r e o f the Ribonuclease H

SMITH MC, COOK JA, FURMAN TC, GESELLCHEN PD, SMITH DP, HSlUNG H: C h e l a t i n g P e p t i d e - l r n m o b i l l z e d M e t a l - i o n A f f i n i t y C h r o m a t o g r a p h y . In Protein Purification. Edited by Ladisch MR, WiUson RC, Painton CC, Builder SE. Washington DC: American Chemical Society 1990, 427:168-180.

43.

HIGAKIJN, FLETTEVaCK RJ, CRAIK CS: E n g i n e e r e d Metall o r e g u l a t i o n i n E n z y t n e s . Trends in Biochem Sci 1991, 17:100-104. A review of e n g i n e e r e d metal sites for the regulation of e n z y m e activity is presented. 44.

TOMA S, CAMPAGNOL S, MARGARIT I, GIANNA R, GRANDI G, BOLOGNESI M, DE FILIPPIS V, FONTANA A: G r a f t i n g o f a C a l c i u n a - b i n d i n g L o o p o f T h e r m o l y s i n to B a c i l l u s subtilis Neutral P r o t e a s e . Biochemistry 1991, 30:97-106. Replacing a loop o f neutral protease with a Ca-binding loop of thermolysin provided a mutant protein with one more Ca-binding site than the wild-type protein. Binding was m e a s u r e d by atomic absorption spectroscopy. Ca(II) modulation of e n z y m e stability in the mutant correlates with similar findings previously reported for thermolysin. 45. ,.

LU Y, GRALLA EB, ROE JA, VALENTINEJS: R e d e s i g n o f a T y p e 2 i n t o a T y p e 1 C o p p e r Protein: Construction and Characterization o f Yeast Copper-zinc S u p e r o x i d e D i s m u t a s e Mutants. J A m Chem Soc 1992, in press. A single mutation of a His to Cys in the Zn-binding site of the enzyme superoxide dismutase created a Cu-binding site with spectral characteristics of a type I Cu site. This is the first successful design of a type I site. 46. •,

HELLINGA HW, RICHARDS FM: C o n s t r u c t i o n o f N e w Liga n d B i n d i n g Sites i n P r o t e i n s o f K n o w n S t r u c t u r e . L Computer-aided M o d e l i n g o f Sites w i t h P r e d e f i n e d Geo m e t r y . J Mol Biol 1991, 222:763-785. The computer p r o g r a m DEZYMER has b e e n developed to build n e w ligand-binding sites into a protein of k n o w n three-dimensional structure. The p r o g r a m searches for b a c k b o n e positions that are appropriate for providing reasonable side chain geometry for ligand binding. HELLINGAHXX?, CARADONNAJP, RICHARDS FM: Construction o f N e w L i g a n d B i n d i n g Sites i n P r o t e i n s o f K n o w n Structure. II. G r a f t i n g o f a B u r i e d T r a n s i t i o n Metal B i n d i n g Site into Escherichia coli T h i o r e d o x i n . J Mol Biol 1991, 222:787-803. A potential type I Cu-binding site predicted by the program DEZYMER w a s built into the protein thioredoxin. Although Hg(II) is coordinated in the predicted manner, Cu(II) does not bind in the w a y predicted by the original design. 47. •.

Protein metal-binding sites Tainer, Roberts, Getzoff 387 48.

LIEBERMANM, SASAKI T: I r o n ( H ) O r g a n i z e s a Synthetic Peptide i n t o T h r e e - h e l i x B u n d l e s . J A m Chem Soc 1991, 113:1470-1471. The addition of Fe(II) to 15-residue peptides containing N-terminal bipyridine groups provided a three-helix bundle showing greater than 85% helicity by circular dichroism spectroscopy. 49.

GHADIRIMR, SOARES C, CHOI C: A C o n v e r g e n t A p p r o a c h to Protein Design. Metal Ion-assisted S p o n t a n e o u s Selfa s s e m b l y o f a Polypeptide i n t o a T r i p l e - h e l i x Bundle P r o t e i n . J A m Chem Soc 1992, 114:825-831. Three helix-bundles resulted from addition of Ni, Co, or Ru to 15residue peptides with bipyridine groups. 50. •,

GHADIPa MR, SOARES C, CHOI C: D e s i g n o f a n Artificial F o u r - h e l i x B u n d l e M e t a i l o p r o t e i n v i a a N o v e l Rutheniuam(II)-assisted Self-assembly Process. J A m Chem Soc 1992, 114:4000-4002. A four-helix bundle m a d e of four 15-amino-acid peptides, each having a N-terminal pyridyl group, undergoes a novel intermolecular self-assembly process u p o n Ru(ID complexation. Spectroscopic evidence indicates greater than 90% helicity.

51. •.

RUANF, CHEN Y, ITOH K, SASAKI T, HOPKINS PB: Synthes i s o f Peptides C o n t a i n i n g U n n a t u r a l , Metal-ligating Residues: Aminodiacetic Acid a s a Peptide Side C h a i n . J org Chem 1991, 56:4347-4354. Peptides consisting of a mixture of a m i n o acids and residues with aminodiacetic acid side chains were synthesized. Addition of metal ions cause appreciable e n h a n c e m e n t of the helix content of some of these peptides. 52.

TAINER JA, ROBERTS VA: E n z y m e Motifs i n A n t i b o d i e s . Nature 1990, 348:589.

53.

KRAULIS PJ: MOLSCRIPT: A Progratn to Produce Both Detailed and Schematic Plots o f Protein Structures. J Appl Cryst 1991, 24:946-950.

JA Tainer, VA Roberts a n d ED Getzoff, Department of Molecular Biology, MB4, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA.