Domain motions in proteins

Domain motions in proteins Georg E. Schulz Institut for Organische C h e m i e u n d Biochemie der Universit~t, Freiburg im Breisgau, G e r m a n y Although proteins require and possess well defined spatial structures, ever more cases are emerging where parts of proteins have moved relative to each other. These parts can be as small as single side chains or as large as domains of 50-150 residues. Analysis of these motions yields valuable data on the functions of the respective proteins. Current Opinion in Structural Biology 1991, 1:883-888

Introduction At present a large number of protein structures and even more amino acid sequences have been recorded. From these data, it has become clear that protein architecture is based on a modular system in which domains of 50-150 residues are the building blocks. (The best known example of this is the domain structure of antibodies.) The domains are most likely separate folding units. They consist of integral chain segments that are arranged like pearls on a string and have been frequently exchanged between proteins during the evolutionary process of protein differentiation. A typical example of a modular enzyme is glutathione reductase, for which several domains with defined simple functions (FAD binding, NADPH binding and interface formation) combine to catalyze a rather complicated chemical reaction. Where domains are the building blocks, one might expect that conformational changes within proteins will in the first place be motions of these domains relative to each other. Recent results satisfy this expectation to a certain extent. In several cases, however, the hinge regions between moving parts are strengthened by additional chain connections. This implies that the moving unit consists of several chain segments that are not consecutive along the polypeptide chain. It is a matter for discussion whether or not such a moving unit should also be denoted as a domain.

Domain rearrangements during evolution Protein differentiation has worked in such a way as to produce families of proteins that share either a single domain or an assembly of domains. An example for proteins with a single resembling domain is given by the flavoenzyme group: cholesterol oxidase [1], p-hydroxybenzoate hydroxylase and glutathione reductase. Multiple domain similarities are, for instance, found in the flavoenzyme group: lipoamide dehydrogenase, mercuric ion reductase [2], NADH peroxidase, thioredoxin reductase [3°], trypanothione reductase and glutathione reductase. When the structures of multidomain proteins are

compared, one expects to find domain rearrangements, which are usually quantified by superpositions of corresponding Ca atoms. Experiences with glutathione reductase have shown that changes are mostly rearrangements of domains [4,5]. Moreover, they are consistent with the original visual domain assignment from electron-density maps: two domains (the FAD-binding and central domains) that are well separated along the chain but have a large and tight common interface move together. The concomitant motion of these two domains over an angle as large as 66* relative to the NADP domain has also been reported in a comparison of thioredoxin reductase with glutathione reductase [3"]. These rearrangements confirm the modular architecture of larger proteins even better than sequence and structure similarities.

Motions during functional cycles Numerous proteins have been structurally analyzed in their unliganded and liganded forms. Because conformational changes upon ligand binding can generally be related to protein function, these analyses improve our knowledge of the mechanical motions within proteins appreciably. The observed changes may involve any number of residues; in this review, the discussion is concemed with motions of more than about a dozen adjacent residues.

Motions in transport proteins The structures of proteins that carry small solutes through the periplasm of Gram-negative bacteria have been studied intensely; with the recent report of the maltose-binding protein [6"], seven such structures are now established. All of these proteins have two lobes, each consisting of more than 100 residues. In five cases, the 'closed' form is observed in which the respective ligand is located between closely associated lobes. In two cases, no ligand is bound and the lobes are 18A apart (relative rotation is 30*) giving rise to a broad cleft [7]. This 'open' form seems to be intrinsically stable as it is

O Current Biology Ltd ISSN 0959-440X

883

884

Proteins identical for two homologous proteins (80 % sequence identity) in different crystal packing environments [8]. Obviously, these transport protons possess two stable conformations and llgand binding and release involves considerable motions of large polypeptide masses. After the structures of the Fe3+-transporting serum protein lactoferrin in man and in rabbit had been elucidated [9"], it became clear that an appreciable conformational change is required for the release of Fe3 +, which is deeply buried between two lobes of about 150 residues each. The structure of human apolactoferrin has since become available and shows that in the absence of Fe3 + (and the concomitantly bound carbonate) a broad cleft has appeared between the two lobes [ 10.o ]. The motion can be described as a rigid body rotation of one of the lobes (rotation angle 53 °) giving rise to Cccatom shifts of up to ,-, 20A. This conformational change is of the same magnitude as that observed for the periplasmic transport proteins [7]. The large structural difference between apoand hololactoferrin had been detected earlier as considerable gyration radii differences in small angle X-ray scattering experiments [11]. A much less dramatic polypeptide movement has recently been reported for annexin-V, which participates in Ca2 + translocation through membranes [12.]. Annexin-V consists of two lobes, each of ,-~ 150 residues [13], that rotate by 4* against each other after binding several Ca 2 + ions (the largest Ca shift is about 2/~). Within the limits of error this is a rigid body motion.

Motions in protein regulation The regulation of O2-binding properties by large changes of quaternary structure accompanied by smaller changes in tertiary structure are well known for deoxy- and oxyhaemoglobin, where the subunits 0tl[31 undergo a rotation of 15° relative to subunits 0t2~2 [14"]. A similar dramatic difference between two regulatory states has recently been described for tetrameric (D 2 symmetry) fructose-l,6-bisphosphatase, where the relative modons of the subunit pairs amounts to 19° [15"]. The change is probably coupled to the binding of the allosteric inhibitor AMP. Similar quatemary changes had been reported between the R- and T-states of hexameric (D 3 symmetry) aspartate carbamoyltransferase [16.]. The analysis of these changes has recently been improved by structure analyses of the CTP- and ATP-liganded enzyme in the R- and T-states which showed small differences between the CTP- and ATP-liganded species [16%17-]. It seems reasonable to discuss these quaternary motions together with the domain motions because the distinction between a quaternary structure composed of singledomain subunits and the tertiary structure of a multidomain p r o t o n is fuzzy. Functional recombinant proteins are frequently produced in both forms. Motions affecting regulation in monomeric enzymes have recently been reported for lipases [18,19,20-']. The activation of these lipases by contact with nonpolar surfaces could not be explained from the structures because they failed to show non-polar protein

surfaces [18,19]. Recently, the structure of a lipase in complex with a triacylglyceride analogue was solved and compared with the structure of the unliganded form, revealing a substantial movement of about 10 residues [20.-]. Analogue binding to the active center had caused the displacement of an amphiphilic 0c-helix (rotation angle 167 °, maximum CoL shift 8A) which had covered the catalytic center of the enzyme and thus had inhibited it. The or-helix rolls like a rigid cylinder over the proton surface, opens the catalytic center and covers an adjacent polar surface, presenting its own non-polar surface to the solvent. Given this conformational transition, it is quite likely that the presentation of a non-polar surface entices the enzyme to assume its active state, which has a non-polar counter-surface for binding. This explains the activation riddle.

Motions durin 8 enzyme catalysis Numerous reports exist of small and local conformational changes induced by ligand binding to enzymes. Most of these reports are based on soaking experiments with enzyme crystals of known structure that can usually be performed easily. In contrast, large conformational changes generally break the crystals upon soaking, and often require tedious new crystallizations and structure

analyses. The first enzyme to be shown to experience a large change was hexokinase, which was found in an unliganded 'open' form and in a 'closed' form containing the bound substrate glucose [21]. The structural difference can be described as a rigid body rotation of 12 ° of a lobe of more than 100 residues with Ca shifts up to 8A. A corresponding observation was made for citrate synthase in which a lobe of ,,-95 residues undergoes a rigid body rotation of 18 ° (maximum C~ shift ~ 123,) upon binding coenzyme A [22]. Similar motions were expected for phosphoglycerate kinase where the structures of two species showed that the enzyme consists of two domains separated by a wide cleft. This cleft is known to accomodate the substrates, but is too large to allow phosphoryt transfer. Accordingly, the observed conformation is considered as the open form and a search for the closed form has begun. A recent NMR analysis has contributed to this search [23]. It showed that a mutation in the hinge region between the domains affects substrate binding affinity, presumably by changing the native pattern of domain motions. In adenyiate kinases, large conformational changes were expected for a long time, because all kinases require an induced-fit to prevent phosphoryt transfer to water (ATP hydrolysis), and adenylate kinase is the smallest of them all. For some time, an unliganded structure of this enzyme was known, which showed a large cleft and could be considered as the open form. Recently, structures of this enzyme were solved with one substrate (AMP) bound [24] and with both substrates (AMP and ATP) bound [25]. The analyses showed conformations that were haft-closed after binding AMP and fully closed after binding both substrates. A comparison of the open,

Domain motions in proteins Schulz

half-closed and closed forms revealed very large motions of the two polypeptide domains [26-.], as illustrated in Fig. 1. On binding AMP alone, a domain of 30 residues rotates by 24* (maximum Cot shift 8A). Binding of AMP and ATP causes the same domain to undergo a further 18" rotation with Ca shifts of up to 8A. The resulting overall rotation angle of this domain between open and closed states is 39* (maximum Ca shift 13A). These motions cannot be accurately described as rigid body rotations because they obviously contain shearing components. A much larger motion is observed for a second domain of 38 residues, which is named 'Insert' because it is small (11 residues) in eukaryotic adenylate kinases but consistently large (38 residues) in prokaryotic species. On binding both substrates this domain rotates as a rigid body by 92", giving rise to a maximum Ca shift of 32 A. Whereas the unliganded enzyme (Fig. la) has a spatially extended conformation, the fully liganded form is a rather globular entity (Fig. ld). A detailed analysis of the change showed that

,+AMP~>

A

_7-

+ATP

Shorter movements involving 38 residues per monomer (maximum Ca shift ~ 6 A ) have been observed with the human immunodeficiency virus protease on binding its inhibitor acetyl-pepstatin, a pentapeptide [29°]. Interestingly, no movements occurred on binding a specially designed octapeptide inhibitor [30°]. Mobility within this

.

(d)

(c)

the catalytic center is disassembled in the open and halfclosed forms and only assembled upon full closure. The enzyme really takes precautions to avoid unintentional ATP hydrolysis. In another analysis a closed form in addition to a known open form has recently been described for aspartate amino transferase [27°]. Upon binding substrate analogues, a lobe of ,,~ 120 residues moves down and closes the active center• The motion is a rigid body rotation of about 13" with a maximum Ca shift of ,-,7A, The motion of a smaller chain segment of only 11 residues has been reported for triosephosphate isomerase [28], where a flexible loop closes down on the bound substrate (maximum Ca shift is 7A).

Fig. 1. Domain motions correlated with substrate binding in adenylate kinases. (a) 'Open' structures of eukaryotic cytosolic adenylate kinase poised to accept substrates. (b) Mitochondrial adenylate kinase with bound substrate AMP superimposed on the cytosolic enzyme but laterally displaced. A lobe of 30 residues (the AMP-binding domain) has moved to yield a 'half-closed' form. (c) Same as (b) but rotated by 90" around a vertical axis. (d) Bacterial adenylate kinase with bound substrates ATP and AMP (inhibitor ApsA) superimposed on (c) but laterally displaced. A lobe of 38 residues (the 'Insert domain') at the top of the molecule moves downwards (rotation 92°, C~ shift up to 32 A) to close the catalytic center and form a 'closed' globular protein. In addition, the AMP-binding domain has closed up further.

885

886

Proteins

protease was also the subject of a recent computational analysis [31], in which the most mobile portions were derived from the trajectories of a long molecular dynamics simulation. The simulation indicated motions of the 'flaps' at the active center and neighboring chain parts that were indeed observed on binding acetyl-pepstatin [29"]. A detailed analysis of the correlation between experiment and simulation would be of interest. More indirect data on domain motions during catalysis have been obtained from the structure analysis of mutant Met6--+Ile of T4 lysozyme [32 -o] which crystallized with four independent molecules in the asymmetric unit. As T4 lysozyme consists of two domains that form a large cleft for substrate binding, domain rotation around a hinge at the cleft has been discussed for a long time. The mutation Met6--+Ile at this hinge obviously softens the local structure, facilitating rotations and giving rise to the concomitant crystallization of four molecules in different rotational states: 9", 26", 27~ and 32* away from the standard conformation (maximum Ca shift 9 •). This example lacks a direct connection with substrate binding but demonstrates the available conformational space for domain motions. The hinge bending motion of the homologous human lysozyme was the subject of a recent computational normal mode analysis [33"], which confirmed the general expectation that the lowest frequency modes contribute most strongly to hinge bending. One should keep in mind, however, that such an analysis is severely impeded by the assumption of harmonic potentials allowing only small rotations of the order of 3 °.

Conclusions

Recent advances have facilitated X-ray structure analyses of protein crystals, increasing the rate of appearance of new structures. Among these, more and more structures of slightly different proteins have become available and have stimulated comparisons for exploring structure-function relationships. The observed differences have either evolved over the long history of protein differentiation, or are caused by designed mutations, or display different functional states of a single protein. For all large motions within functional cycles, it turns out that the moving peptide entities are connected by more than one chain segment to the bulk protein. Thus, each motion requires softening of a hinge region, i.e. more than a simple rotation of a main chain dihedral angle. Presumably, only a spacious hinge can restrict a protein to a few, or even two, conformations. Mutations at the hinge cause disturbances leading to a multitude of conformational states in T4 lysozyme [32"-] and to a change of these states in phosphoglycerate kinase [23]. This observation contrasts with the situation found in antibodies where the hinges between the Fab and Fc segments consist only of single chains and allow the random motions that are necessary for the function of antibodies. It should be mentioned that most reported data were derived from crystals, where the crystallization process can

enforce one suitable packing conformation. It is therefore advisable that deviating conformations are established in more than one crystal-packing arrangement before they are considered as intrinsically stable. A possible packing effect has been suggested, for instance, for the small domain movements in annexin-V [12.]. The large motions in the periplasmic transport proteins [7,8] and in adenylate kinases [26-] have been confirmed for several crystal-packing arrangements.

References and recommended reading Papers of special interest, published within the annual period of review,

have been highlightedas: • •.

of interest of outstanding interest

1.

VPaEUNKA, LtOYD LF, BroW DM: Crystal Structure of Cholesterol Oxidase from Brevibactertum steroltcum Refined at 1.8A Resolution. J Mo/Bto/1991, 219:533-554.

2.

SCHIERINGN, KABSCHW, MOOREMJ, DISTEFANOMD, WAtSH CT, PAl EF: Structure of the Detoxilicatioo Catalyst Mercuric Ion Reductase from Bacillus sp Strain RC607. Nature 1991, 352:168-171.

3. •

KtmIYANJ, KRISHNA TSR, WONG L, GUENTHER B, PAHLER A, WuaaAMSCH JR, MODEL P: Convergent Evolution of Similar Function in Two Structurally Divergent Enzymes. Nature 1991, 352:172-174. TMoredoxin reductase has a peculiar relationship to the other disulphide oxidoreductases (exemplified by glutathione reductase). It lacks the interface domain, it is monomeric, there is a gross rearrangement between its NADP-binding and FAD-binding/central domains and its disulfide bond is at the other side of the isoaUoxazine of FAD. The crystalline enzyme is an inactive mutant. 4.

SCHULZGE, ERMLERU: Structural Changes on Binding FAD and FAD-analogues and o n Site-directed Mutagenesis of Glutathione Reductase. In Flavins and Flavoproteins 1990 edited by Curd B, Ronchi S, Zanetti G [book]. Berlin: de Gruyter, 1991, pp 505-512.

5.

ERMLERE, SCI4ULZGE: The Three-dimensional Structure of Glutathione Reductase from E$cherlchia coil at 3.0 It Resolution. Proteins 1991, 9:174-179.

6. ,

SPURLtNOJC, LU G-Y, QUIOCHO FA: The 2.3Jr Resolution Structure of the Maltose- or Maltodextrin.binding Protein, a Primary Receptor of Bacterial Active Transport and Chemotaxis. J Biol Chem 1991, 266:5202-5219. The structure described is the seventh to be reported for a periplasmic binding protein. Although the seven structures are generally similar, the chain folds deviate considerably in detail. 7.

SACKJS, SAPERMA, QUIOCHO FA: Periplasmic Binding Protein Structure and Function. Refined X-ray Structures o f the Leucine/Isoleucine/Valine-binding Protein and its Complex with Leucine. J Mol Biol 1989, 206:171-191.

8.

SACKJS, TRAKHANOVSD, TSIGANNIKIll, QUIOCHO FA~ Structure of the L-Leucine-binding Protein Refined at 2.4-A Resolution and Comparison with the Leu/Ile/Val-binding Protein Structure. J Mol Biol 1989, 206:193-207.

9. .

SARRAR, GARRA~rrR, GOmNSKY B, JHOTI H, LINDLEYP: High-

resolution X-ray Studies on Rabbit Serum Transferrin: Pre-

liminary Structure Analysis o f the N-Terminal Half-molecule at 2.3A Resolution. Acta Crystallogr [B] 1990, 46:763-771. The transferrin fragment consists of only the amino-terminal lobe of the molecule and forms more highly ordered crystals than the whole molecule and, therefore, reveals details of Fe3 + and carbonate binding to the protein.

Domain motions in proteins Schulz 10.

ANDERSONBF, BAKER HM, NORMS GE, RUMBALLSV, BAKER EN: Apolactoferrin Structure Demonstrates ligand-induced Conformational Change in Transferrins. Nature 1990, 344:784--787. Lactoferrin binds two Fe2 + ions, one at its amino-terminal and one at its carboxy-terminal lobe. These two lobes are structurally similar. Apolactoferrin lacks both Fe2 + ions and shows a large rearrangement in the amino-terminal lobe but no gross change in the carboxy-terminal lobe. This differential behaviour may be caused by crystal packing forces.

PATKARSA, THIM Iz A Model for Interfacial Activation in Lipases from the Structure of a Fungal Lipase--Inhibitor Complex. Nature 1991, 351:491-494. Both reported triacylglycerol lipases [ 19,20 *•] contain a buried catalytic Ser...His... Asp triad similar to that found in the serine proteases. Activation of these enzymes by non-polar surfaces remained a riddle until analogue binding to one of the lipases showed that a non-polar surface patch developed adjacent to the solvent-accessible catalytic triad after an appreciable conformational change.

11.

~ F, SIMON I: The Effect of Iron Binding on the Conformation of Transferrin. A Small Angle X-Ray Scattering Study. n ~ o b y s J 1985, 48:799-802.

21.

BENNETtWS JR, STErlZ T& Glucose-induced Conformational Change in Yeast Hexokinase. Proc N a a Acad Sci USA 1978, 75:4848-4852.

HUBER R, SCHNEIDER M, MAYR I, ROMISCH J, PAQUES E-P: The Calcium Binding Sites in Human Annexin V by Crystal Structure Analysis at 2.0A Resolution. FEBS Left 1990, 275:15-21. The structure of a second crystal form of annexin-V is reported that allows Ca2+ binding upon soaking. This crystal form shows a small rotation of one lobe relative to the other which could be of functional importance. However, a crystal-packing effect could also explain this movement.

22.

REMINGTONS, WIEGAND G, HUBER Pc Crystallographic Refinement and Atomic Models of Two Different Forms of Citrate Synthase at 2.7 and 1.7A Resolution. J Mo/B/o/1982, 158:111-152.

23.

GRAHAMHC, WIIAJ.M~ RJP, LITrLECHILD JA, WATSON HC: A Proton-NMR Study o f a Site-directed Mutation (His388--* Glu) in the Interdomain Region o f Yeast Phosphoglycerate Kinase. Implications for Domain Movement. Eur J Biocloem 1991, 196".261-269.

13.

HUBERP,, ROMISCHJ, PAQUES E-P: The Crystal and Molecular Structure of Human Annexin V, an Anticoagulant protein that Binds to Calcium and Membranes. EMBO J 1990, 9:3867-3874.

24.

DmDEPaCHSK, SCHULZGE: The Relined Structure of the Complex Between Adenyiate Kinase from Beef Heart Mitochondrial Matrix and its Substrate AMP at 1.85 A Resolution. J Mo/Bt~d 1991, 217:541-549.

SMrlH FR, LATrMAN EE, CARTER CW JR: The Mutation [~99 Asp-Tyr Stabilizes Y - - a New, Composite Quaternary state of Human Hemoglobin. Proteins 1991, 10:81-91. The carbonmonoxy form of this mutant haemoglobin shows a quaternary structure that differs appreciably from that of the deoxy- and oxy-hemoglobin structures. The mutation occurs within the subunit interface region, which is considered important for the deoxy-oxy switch.

25.

MUELLERCW, SCHUIZ GE: Structure of the Complex of Adenylate Kinase from Escherlchta coIt w i t h the In. hibitor P1,ps-di(adenosine-5'-)pentaphosphate. J Mo/ Bt~0/ 1988, 202:909-912.

**

12. .

14.

.

15. .

KE H, ZHANGY, LIPSCOMBWN: Crystal Structure of Fructose1,6-bisphosphatase Complexed with Fructose-6-phosphate, AMP, and Magnesium. Proc Natl Acad Sci USA 1990, 87:5243-5247. The structure of the enzyme complexed with fructose-6-phosphate, AMP and Mg2+ differs grossly from the structure with bound fructose2,6-bisphosphate, which together with AMP regulates the activity of the enzyme. The differences are mostly restricted to the tetrameric quaternary structure; one pair of subunits rotates relative to another. 16. .

GOUAUXJE, STEVENSRC, LtPSCOMBWN: Crystal Structures of Aspartate Carbamoyltransferase Ligated With Phosphonoacetamide, Malonate, and CTP or ATP at 2.8 A Resolution and Neutral pH. B~.hemistry 1990, 29:7702-7715. These two aspartate carbamoyttransferase papers [16%17 •] report several stuctures of the R- and T-states of the enzyme with bound regulators ATP (activator) and CTP (inhibitor). The structural differences between the complexes with ATP and CTP are rather small. In contrast, there are hrge quaternary structure changes (rotations around a symmetry axis of up to 15°, relative subunit shifts of up to 12A) between the R- and T-states. 17. •

STEVENSRC, GOUAUX JE, LIPSCOMB WN: Structural Consequences of Effector Binding to the T State of Aspartate Carbamoyltransferase: Crystal Structures of the Unliganded and ATP- and CTP-complexed Enzymes at 2.6A Resolution. Bio6bemistry 1990, 29:7691-7701. See [16*] 18.

BRADYL, BRZOZOWSKIAM, DEREWENDAZS, DODSON E, DODSON G, TOI.LEY S, TURKENBURGJP, CHRISTIANSEN L, HUGE-JENSEN B, NORSKOV L, THIM L, MENGE U: A Scrine Proteasc Triad Forms the Catalytic Centre of a Triacylgiycerol Lipase. Nature 1990, 343:767-770.

19.

Wfi~d.ERFK, D'ARCY A, HUNZIKER W: Structure of Human Pancreatic Lipasc. Nature 1990, 343:771-774.

20.

BRZOZOWSKIAM, DEREWENDAU, DEREWENDAZS, DODSON GG, LAWSON DM, TURKENBURGJP, BJORKLING F, HUGE-JENSEN B,

**

26. SCHULZGE, MUELLERCW, DIEDERICHS K: Induced-lit Move** ments in Adenylate Kinases. J Mol Biol 1990, 213:627-630. The three structures compared are confirmed as intrinsically stable because they occur in different crystal-packing arrangements: the 'open' form is known from two different packings of eukaryotic enzymes; the 'half-closed' form exists as two crystaUographically independent molecules in the asymmetric unit (i.e. in different packing arrangements); and, the 'closed' form is observed in three enzyme-ApsA complexes in two crystal forms (ApsA is p1, p5.bis(adenosine.5,)pentaphos. phate, it mimicks the two substrates ATP and AMP), one of which contains two independent molecules in the asymmetric unit. Accordingly, crystal packing arefacts are unlikely and the comparison is well founded. 27. .

PICOT D, SANDMEIER E, THALLER C, VINCENT MG, CHRISTEN P, JANSONIUSJN: The Open/Closed Conformational Equilibrium of Aspartate Aminotransferase. Studies in the Crystalline State and with a Fluorescent Probe in Solution. Eur J B/ochem 1991, 196:329-341. An analysis of two crystal forms at a rather low resolution of 4.4~, shows that a peptide lobe moves on binding substrate analogues. This motion was followed by analyzing fluorescence changes in solution. Correlation between crystal and solution studies is reported. 28.

JOSEPHD, PETSKO GA, KARPLUSM: Anatomy of a Conformational Change: Hinged 'Lid' Motion of the Trioscphosphate Isomerase Loop. Science 1990, 249:1425-1428.

29.

FITZGERALD P i n , MCKEEVER BM, VANMIDDLESWORTH JF, SPRINGERJP, HEIMBACH JC, LEU C-T, HERBER WK, DIXON RAF, DARKE Pk Crystallographic Analysis of a Complex Bet w e e n Human Immunodeficiency Virus Type 1 Protease and Acetylpepstatin at 2.0A Resolution. J Bkd ~ 1990, 265:14209--14219. Because of its medical importance, this protease enjoys a very high general interest. An intense search for inhibitors is under way. The structure is reported with the peptide inhibitor acetyl-pepstatin which induces an appreciable conformational change. .

30. ,

JASKOLSKI M, TOMASSELLI AG, SAWYER TK, STAPLES DG, HEINRIKSONRIo SCHNEIDERJ, KENT SBH, WLODAWERA: Structure at 2.5A Resolution o f Chemically Synthesized Human Immunodeficiency Virus Type 1 Protease Complexed with a Hydroxyethylene-based Inhibitor. Biochemistry 1991, 30:1600-1609.

887

888

Proteins The structure is reported as ligated with a peptide inhibitor different from the one used in [29•]. In contrast to [29•], only minor conformational changes are observed. 31.

32. ••

HARTE WE JR, SWAMINATHAN S, MANSURI MM, MARTIN JC, ROSENBERG IE, BEVERIDGEDL: Domain Communication in the Dynamical Structure of H u m a n Immunodeficiency Virus 1 Protease. Proc Natl A c a d Sci USA 1990, 87:8864-8868.

FABER HR, MATrHEWS BW: A Mutant I"4 Lysozyme Displays Five Different Crystal Conformations. Nature 1990, 348:263--266. Mutants ofT4 lysozyme have been studied by this group for quite some time. Up to now, these analyses have been mostly restricted to small changes that did not change the crystal form, keeping the analyses easy. The more tedious elucidation of a new crystal form of a mutant has yielded most interesting information on the nature of the hinge, which appears to restrict the enzyme to only a few stable conformations. Destroying the hinge by mutation allows for a multitude of intermediate states.

33. •

GIBRATJ-F, Go N: Normal Mode Analysis of H u m a n Lysozyme: Study of the Relative Motion o f t h e T w o Domains and Characterization of the Harmonic Motion. P r ~ teins 1990, 8:258-279. It has long been expected that there exists a hinge-bending vibrational mode in lysozyme which, as a result of the large masses involved, should have a low frequency. The study confirms this, the free lowest frequency modes can be combined to model hinge bending. Being in vacuum and restricted to harmonic potentials, however, the analysis cannot contribute much to the analysis of the actual hinge bending, as it does not apply to the natural environment and deals with only minor vibrational amplitudes.

GE Schulz, lnstitut for Organische Chemie und Biochemie der Universi~t, Albertstrasse 21, 7800 Freiburg im Breisgau, Germany.