Elastic and inelastic diffraction changes upon variation of the relative humidity environment of PurE crystals.

The different changes observed in the diffraction patterns of three different crystal forms (hexagonal, trigonal and monoclinic) of PurE (EC 4.1.1.21), an enzyme from the purine-biosynthesis pathway of Bacillus anthracis, upon a wide range of changes in the relative humidity environment of the crystals are documented. In addition, the changes in the unit-cell parameters, volume and bulk solvent in the three different crystal forms were systematically followed. In an attempt to explain the elastic (P6(5)22) and inelastic (P3(1)21) changes in the diffraction pattern, refined structures of the three different crystal forms determined at 100 K are presented, with particular emphasis on the tertiary and quaternary structural differences, crystal packing, intermolecular and intramolecular interactions and solvent structure. The refined structures show that the precipitant salts, solvent structure (both ordered and bulk) and conformation of the C-termini all play a role in creating a unique cement at both the intramolecular and intermolecular contacts of the different crystal forms. It is suggested that it is the combination of polyethylene glycol and the structure of the ordered water molecules (first and second layers) as well as the structure of the bulk solvent that are the critical factors in the plasticity of the hexagonal crystal packing as opposed to the inelastic responses of the lower symmetry forms.

Humidity control can compensate for the damage induced in protein crystals by alien solutions.

The use of relative humidity control of protein crystals to overcome some of the shortcomings of soaking ligands (i.e. inhibitors, substrate analogs, weak ligands) into pre-grown apoprotein crystals has been explored. Crystals of PurE (EC 4.1.1.21), an enzyme from the purine-biosynthesis pathway of Bacillus anthracis, were used as a test case. The findings can be summarized as follows: (i) using humidity control, it is possible to improve/optimize the diffraction quality of crystals soaked in solutions of organic solvent (DMSO, ethanol) containing ligands/inhibitors; (ii) optimization of the relative humidity can compensate for the deterioration of the diffraction pattern that is observed upon desalting crystals grown in high salt; (iii) combining desalting protocols with the addition of PEG it is possible to achieve very high concentrations of weak ligands (in the 5-10 mM range) in soaking solutions and (iv) fine control of the relative humidity of crystals soaked in these solutions can compensate for the deterioration of crystal diffraction and restore `high-resolution' diffraction for structure-based and fragment-based drug design. It is suggested that these experimental protocols may be useful in other protein systems and may be applicable in academic or private research to increase the probability of obtaining structures of protein-ligand complexes at high resolution.

Candida proteases and their inhibition: prospects for antifungal therapy.

The rationale for inhibition of the secreted acid proteases (SAP) of Candida fungal species as a novel antifungal strategy is outlined. Enzyme structure-activity relationship data regarding the inhibitor A-70450 are described along with results from in vivo antifungal assays. Developments from protein X-ray crystallographic studies, SAP knock-out genetic studies, and the importance of these new results for drug discovery are reviewed. Finally, inhibition of the Candida SAP enzymes with HIV protease inhibitors and a proposed role in candidiasis of AIDS patients is discussed.

Crystal structure of Lysbeta(1)82-Lysbeta(2)82 crosslinked hemoglobin: a possible allosteric intermediate.

The crystal structure of human hemoglobin crosslinked between the Lysbeta82 residues has been determined at 2.30 A resolution. The crosslinking reaction was performed under oxy conditions using bis(3, 5-dibromosalicyl) fumarate; the modified hemoglobin has increased oxygen affinity and lacks cooperativity. Since the crystallization occurred under deoxy conditions, the resulting structure displays conformational characteristics of both the (oxy) R and the (deoxy) T-states. beta82XLHbA does not fully reach its T-state conformation due to the presence of the crosslink. The R-state-like characteristics of deoxy beta82XLHbA include the position of the distal Hisbeta63 (E7) residue, indicating a possible reason for the high oxygen affinity of this derivative. Other areas of the molecule, particularly those thought to be important in the allosteric transition, such as Tyrbeta145 (HC2) and the switch region involving Proalpha(1)44 (CD2), Thralpha(1)41 (C6) and Hisbeta(2)97 (FG4), are in intermediate positions between the R and T-states. Thus, the structure may represent a stabilized intermediate in the allosteric transition of hemoglobin.

The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism.

The rRNA methyltransferase ErmC' transfers methyl groups from S -adenosyl-l-methionine to atom N6 of an adenine base within the peptidyltransferase loop of 23 S rRNA, thus conferring antibiotic resistance against a number of macrolide antibiotics. The crystal structures of ErmC' and of its complexes with the cofactor S -adenosyl-l-methionine, the reaction product S-adenosyl-l-homocysteine and the methyltransferase inhibitor Sinefungin, respectively, show that the enzyme undergoes small conformational changes upon ligand binding. Overall, the ligand molecules bind to the protein in a similar mode as observed for other methyltransferases. Small differences between the binding of the amino acid parts of the different ligands are correlated with differences in their chemical structure. A model for the transition-state based on the atomic details of the active site is consistent with a one-step methyl-transfer mechanism and might serve as a first step towards the design of potent Erm inhibitors.

Crystal structure of ErmC', an rRNA methyltransferase which mediates antibiotic resistance in bacteria.

The prevalent mechanism of bacterial resistance to erythromycin and other antibiotics of the macrolide-lincosamide-streptogramin B group (MLS) is methylation of the 23S rRNA component of the 50S subunit in bacterial ribosomes. This sequence-specific methylation is catalyzed by the Erm group of methyltransferases (MTases). They are found in several strains of pathogenic bacteria, and ErmC is the most studied member of this class. The crystal structure of ErmC' (a naturally occurring variant of ErmC) from Bacillus subtilis has been determined at 3.0 A resolution by multiple anomalous diffraction phasing methods. The structure consists of a conserved alpha/beta amino-terminal domain which binds the cofactor S-adenosyl-l-methionine (SAM), followed by a smaller, alpha-helical RNA-recognition domain. The beta-sheet structure of the SAM-binding domain is well-conserved between the DNA, RNA, and small-molecule MTases. However, the C-terminal nucleic acid binding domain differs from the DNA-binding domains of other MTases and is unlike any previously reported RNA-recognition fold. A large, positively charged, concave surface is found at the interface of the N- and C-terminal domains and is proposed to form part of the protein-RNA interaction surface. ErmC' exhibits the conserved structural motifs previously found in the SAM-binding domain of other methyltransferases. A model of SAM bound to ErmC' is presented which is consistent with the motif conservation among MTases.

Structure of secreted aspartic proteinases from Candida. Implications for the design of antifungal agents.

Pathogens of the genus Candida can cause life threatening infections in immuno-compromised patients. The three-dimensional structures of two closely related secreted aspartic proteinases from C. albicans complexed with a potent (Ki = 0.17 nM) inhibitor, and an analogous enzyme from C. tropicalis reveal variations on the classical aspartic proteinase theme that dramatically alter the specificity of this class of enzymes. The novel fungal proteases present: i) an 8 residue insertion near the first disulfide (Cys45-Cys50, pepsin numbering) that results in a broad flap extending towards the active site; ii) a seven residue deletion replacing helix hN2 (Ser110-Tyr114), which enlarges the S3 pocket; iii) a short polar connection between the two rigid body domains that alters their relative orientation and provides certain specificity; and i.v.) an ordered 12 residue addition at the carboxy terminus. The same inhibitor (A-70450) binds in an extended conformation in the two variants of C. albicans protease, and presents a branched structure at the P3 position. However, the conformation of the terminal methylpiperazine ring is different in the two crystals structures. The implications of these findings for the design of potent antifungal agents are discussed.

Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: implications for the design of antifungal agents.

The three-dimensional structure of a secreted aspartic protease from Candida albicans complexed with a potent inhibitor reveals variations on the classical aspartic protease theme that dramatically alter the specificity of this class of enzymes. The structure presents: (1) an 8-residue insertion near the first disulfide (Cys 45-Cys 50, pepsin numbering) that results in a broad flap extending toward the active site; (2) a 7-residue deletion replacing helix hN2 (Ser 110-Tyr 114), which enlarges the S3 pocket; (3) a short polar connection between the two rigid body domains that alters their relative orientation and provides certain specificity; and (4) an ordered 11-residue addition at the carboxy terminus. The inhibitor binds in an extended conformation and presents a branched structure at the P3 position. The implications of these findings for the design of potent antifungal agents are discussed.

Antifungal drug targets: Candida secreted aspartyl protease and fungal wall beta-glucan synthesis.

The incidence of severe, life-threatening fungal infections has increased dramatically over the last decade. Unfortunately, in practice the arsenal of antifungal drugs is limited to flucytosine, a few approved azoles, and polyenes, mainly amphotericin B. This situation is rather precarious in view of the extended spectrum of fungi causing severe disease in immunocompromised patients, development of resistance to some of the currently used agents, and the minimal fungicidal activity of the azoles. Although lagging behind the need for new antifungal agents, the study of fungal biochemistry, physiology, and genetics has undergone a resurgence to new heights of activity, thus providing a framework on which to build drug discovery programs in several new areas, two of which will be discussed in detail: the biology of Candida albicans secreted aspartyl protease with respect to inhibitor discovery, evaluation, and possible clinical utility; and the fungal cell wall beta-glucans with respect to the mechanism and regulation of synthesis and target sites for drug inhibition.

Crystallographic analysis of reversible metal binding observed in a mutant (Asp153-->Gly) of Escherichia coli alkaline phosphatase.

Here we present the refined crystal structures of three different conformational states of the Asp153-->Gly mutant (D153G) of alkaline phosphatase (AP), a metalloenzyme from Escherichia coli. The apo state is induced in the crystal over a 3 month period by metal depletion of the holoenzyme crystals. Subsequently, the metals are reintroduced in the crystalline state in a time-dependent reversible manner without physically damaging the crystals. Two structural intermediates of the holo form based on data from a 2 week (intermediate I) and a 2 month soak (intermediate II) of the apo crystals with Mg2+ and Zn2+ have been identified. The three-dimensional crystal structures of the apo (R = 18.1%), intermediate I (R = 19.5%), and intermediate II (R = 19.9%) of the D153G enzyme have been refined and the corresponding structures analyzed and compared. Large conformational changes that extend from the mutant active site to surface loops, located 20 A away, are observed in the apo structure with respect to the holo structure. The structure of intermediate I shows the recovery of the entire enzyme to an almost native-like conformation, with the exception of residues Asp 51 and Asp 369 in the active site and the surface loop (406-410) which remains partially disordered. In the three-dimensional structure of intermediate II, both Asp 51 and Asp 369 are essentially in a native-like conformation, but the main chain of residues 406-408 within the loop is still not fully ordered. The D153G mutant protein exhibits weak, reversible, time dependent metal binding in solution and in the crystalline state.(ABSTRACT TRUNCATED AT 250 WORDS)

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity.

The substitution of aspartate at position 153 in Escherichia coli alkaline phosphatase by glycine results in a mutant enzyme with 5-fold higher catalytic activity (kcat) but no change in Km at pH 8.0 in 50 mM Tris-HCl. The increased kcat is achieved by a faster release of the phosphate product as a result of the lower phosphate affinity. The mutation also affects Mg2+ binding, resulting in an enzyme with lower metal affinity. The 3-D X-ray structure of the D153G mutant has been refined at 2.5 A to a crystallographic R-factor of 16.2%. An analysis of this structure has revealed that the decreased phosphate affinity is caused by an apparent increase in flexibility of the guanidinium side chain of Arg166 involved in phosphate binding. The mutation of Asp153 to Gly also affects the position of the water ligands of Mg2+, and the loop Gln152-Thr155 is shifted by 0.3 A away from the active site. The weaker Mg2+ binding of the mutant compared with the wild type is caused by an altered coordination sphere in the proximity of the Mg2+ ion, and also by the loss of an electrostatic interaction (Mg2+.COO-Asp153) in the mutant. Its ligands W454 and W455 and hydroxyl of Thr155, involved in the octahedral coordination of the Mg2+ ion, are further apart in the mutant compared with the wild type.

3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity.

Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1) from E. coli has produced an enzyme with increased kcat. The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102. The crystal structure of the Asp101-->Ser mutant has been refined at 2.5 A to a final crystallographic R-factor of 0.173. The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate). The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible. This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.

Structure of a pepsin/renin inhibitor complex reveals a novel crystal packing induced by minor chemical alterations in the inhibitor.

The structure determination by molecular replacement methods of a monoclinic pepsin/renin inhibitor complex crystal, with two molecules in the asymmetric unit, is presented. The atomic model, consisting of two liganded pepsin molecules and 110 water molecules, has been refined to a final crystallographic R value of 0.139 for data between 8 and 2.9 A resolution. The structure reveals a previously undescribed pepsin dimer formed predominantly by polar interactions. Inhibitor binding induces global structural changes in the native enzyme similar, but not identical, to the ones observed in other chemically similar pepsin/renin inhibitor complexes crystallized in an orthorhombic form. A region of the polypeptide chain (residues 292-297) which was not visible in the orthorhombic crystal is well ordered in the presently described structure; possibly induced by crystal contacts. The crystal packing of native pepsin is compared with the two different crystal forms of the inhibited enzyme.

Inhibitor binding induces structural changes in porcine pepsin.

The refined structures of two isomorphous pepsin/inhibitor complexes demonstrate that significant conformational changes take place upon ligand binding for a mammalian representative of the aspartic proteinase family. These differences can be attributed mostly to the concerted rigid body movements of two separate clusters of residues relative to a central core. One cluster in the amino domain comprises the flap, the adjacent beta strand (sheet IV) and helices, as well as the interconnecting loops. The other, larger cluster is in the carboxy end and corresponds approximately to the flexible subdomain described previously. Similar conformational changes are proposed to occur in renin and cathepsin D.

Statistical descriptors for the size and shape of globular proteins.

The atomic structures of proteins epitomize the ideas of complexity and irregularity in three-dimensional objects. For such objects, size and shape are difficult to quantify, and therefore the development of unbiased parameters for these properties could facilitate their description. Statistical analysis of the frequency distribution of interatomic distances in protein structures of different classes has revealed two numerical descriptors that correlate with physicochemical properties of these macromolecules. The median (mu) of the distribution correlates (r greater than .98, n = 45) with variables indicative of size (e.g., molecular weight and radius of gyration). The exponent of the Box-Cox transformation lambda, used for converting this distribution into a symmetrical one, correlated (r = .75, n = 43) with a general dimensionless shape parameter defined as the combination of the shape-related accessible surface (A0s), molecular volume (V), and radius of gyration (Rg) in the form s = (A0sRg/V). It is suggested that for globular proteins lambda is a function of both the shape parameter s and the fractal dimension D of the protein surface. These objective descriptors of size and shape could be useful to describe other complex objects.

Revised 2.3 A structure of porcine pepsin: evidence for a flexible subdomain.

A revised three-dimensional crystal structure of ethanol-inhibited porcine pepsin refined to an R-factor of 0.171 at 2.3 A resolution is presented and compared to the refined structures of the fungal aspartic proteinases: penicillopepsin, rhizopuspepsin, and endothiapepsin. Pepsin is composed of two nearly equal N and C domains related by an intra dyad. The overall polypeptide fold and active site structures are homologous for pepsin and the fungal enzymes. The weak inhibition of pepsin by ethanol can be explained by the presence of one or more ethanol molecules, in the vicinity of the active site carboxylates, which slightly alter the hydrogen-bonding network and which may compete with substrate binding in the active site. Structural superposition analysis showed that the N domains aligned better than the C-domains for pepsin and the fungal aspartic proteinases: 107-140 C alpha pairs aligned to 0.72-0.85 A rms for the N domains; 64-95 C alpha pairs aligned to 0.78-1.03 A rms for the C domains. The major structural difference between pepsin and the fungal enzymes concerns a newly described subdomain whose conformation varies markedly among these enzyme structures. The subdomain in pepsin comprises nearly 100 residues and is composed of two contiguous segments within the C domain (residues 192-212 and 223-299). the subdomain is connected, or "hinged," to a mixed beta-sheet that forms one of the structurally invariant, active site psi-loops. Relative subdomain displacements as large as a 21.0 degrees rotation and a 5.9 A translation were observed among the different enzymes. There is some suggestion in pepsin that the subdomain may be flexible and perhaps plays a structural role in mediating substrate binding, determining the substrate specificity, or in the activation of the zymogen.

Isotope-edited proton NMR study on the structure of a pepsin/inhibitor complex.

A general approach is illustrated for providing detailed structural information on large enzyme/inhibitor complexes using NMR spectroscopy. The method involves the use of isotopically labeled ligands to simplify two-dimensional NOE spectra of large molecular complexes by isotope-editing techniques. With this approach, the backbone and side-chain conformations (at the P2 and P3 sites) of a tightly bound inhibitor of porcine pepsin have been determined. In addition, structural information on the active site of pepsin has been obtained. Due to the sequence homology between porcine pepsin and human renin, this structural information may prove useful for modeling renin/inhibitor complexes with the ultimate goal of designing more effective renin inhibitors. Moreover, this general approach can be applied to study other biological systems of interest such as other enzyme/inhibitor complexes, ligands bound to soluble receptors, and enzyme/substrate interactions.

Refined crystal structure of dogfish M4 apo-lactate dehydrogenase.

The crystal structure of M4 apo-lactate dehydrogenase from the spiny dogfish (Squalus acanthius) was initially refined by a constrained-restrained, and subsequently restrained, least-squares technique. The final structure contained 286 water molecules and two sulfate ions per subunit and gave an R-factor of 0.202 for difraction data between 8.0 and 2.0 A resolution. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.25 A. The elements of secondary structure of the refined protein have not changed from those described previously, except for the appearance of a one-and-a-half turn 3(10) helix immediately after beta J. There is also a short segment of 3(10) helix between beta C and beta D in the part of the chain that connects the two beta alpha beta alpha beta units of the six-stranded parallel sheet (residues Tyr83 to Ala87). Examination of the interactions among the different elements of secondary structure by means of a surface accessibility algorithm supports the four structural clusters in the subunit. The first of the two sulfate ions is in the active site and occupies a cavity near the essential His195. Its nearest protein ligands are Arg171, Asp168 and Asn140. The second sulfate ion is located near the P-axis subunit interface. It is liganded by His188 and Arg173. These two residues are conserved in bacterial lactate dehydrogenase and form part of the fructose 1,6-bisphosphate effector binding site. Two other data sets in which one (collected at pH 7.8) or both (collected at pH 6.0) sulfate ions were replaced by citrate ions were also analyzed. Five cycles of refinement with respect to the pH 6.0 data (25 to 2.8 A resolution) resulted in an R value of 0.191. Only water molecules occupy the subunit boundary anion binding site at pH 7.8. The amino acid sequence was found to be in poor agreement with (2Fobs-Fcalc) electron density maps for the peptide between residues 207 and 211. The original sequence WNALKE was replaced by NVASIK. The essential His195 is hydrogen bonded to Asp168 on one side and Asn140 on the other. The latter residue is part of a turn that contains the only cis peptide bond of the structure at Pro141. The "flexible loop" (residues 97 to 123), which folds down over the active center in ternary complexes of the enzyme with substrate and coenzyme, has a well-defined structure. Analysis of the environment of Tyr237 suggests how its chemical modification inhibits the enzyme.

The structure of rabbit muscle phosphoglucomutase at intermediate resolution.

The three-dimensional structure of rabbit phosphoglucomutase has been determined to 2.7 A resolution by a combination of isomorphous and molecular replacement techniques. Heavy atom positions were found by using vector search and difference Fourier methods. The two molecules in the asymmetric unit form a dimer with its 2-fold axis perpendicular to and intersecting with a crystallographic 4(1) axis. Thus, the dimers are arranged so that they form fibers that are coincident with the 4(1) axes. A polypeptide model, corresponding with the known residue sequence, has been fitted to the electron density map to produce a structure that consists of four domains. All four have an alpha/beta structure; the first three have a somewhat similar topology that is based on a mixed parallel/antiparallel beta sheet, whereas the fourth is based on an antiparallel sheet. The active site lies between the four domains, with the phosphoserine residue in the first domain and some of the probable substrate-binding residues in the fourth and final domain. The carboxyl edges of all four sheets are directed towards the active site region, which lies in a deep crevice.

RNA-protein interactions in some small plant viruses.

The structure of the three quasi-equivalent protein subunits A, B and C of the spherical, T = 3 southern bean mosaic virus (SBMV) have been carefully built in accordance with a refined electron density map of the complete virus. The lower electron density in the RNA portion of the map could not be explicitly interpreted in terms of a preferred RNA structure on which some icosahedral symmetry might have been imposed. However, the extremely basic nature of the interior surface of the coat protein must be associated with the binding and organization of the RNA. Comparison with the small spherical, T = 1 satellite tobacco necrosis virus (STNV; Liljas et al., J. Mol. Biol. 159, 93-108, 1982) and the T = 1 aggregate of alfalfa mosaic virus (AMV) protein (Fukuyama et al., J. Mol. Biol. 150, 33-41, 1981) showed similar results. The pattern of basic residues on the SBMV coat protein surface facing the RNA is able to dock a 9 base pair double-helical A-RNA structure with surprising accuracy. The basic residues are each associated with a different phosphate and the protein can make interactions with five bases in the minor groove. This may be one of a small number of ways in which the RNA interacts with SBMV coat protein. The self-assembly of SBMV has been studied in relation to the presence of the 63 basic amino-terminal coat protein sequence, pH, Ca2+ and Mg2+ ions and RNA. These results have led to a two-state model where the "relaxed" dimers initially self-assemble into 10-mer caps which nucleate the assembly of T = 1 or T = 3 capsids depending on the charge state of the carboxyl group clusters in the subunit contact region. The two-state condition of dimers in a viral coat protein extends the range of structures originally envisaged by Caspar and Klug (Cold Spring Harbor Symp. Quant. Biol. 27, 1-24, 1962).