The 1.9 Å X-ray structure of a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis is reported. This enzyme, MurG, contains two α/β open sheet domains separated by a deep cleft. Structural analysis suggests that the C-terminal domain contains the UDP-GlcNAc binding site while the N-terminal domain contains the acceptor binding site and likely membrane association site. Combined with sequence data from other MurG homologs, this structure provides insight into the residues that are important in substrate binding and catalysis. We have also noted that a conserved region found in many UDP-sugar transferases maps to a β/α/β/α supersecondary structural motif in the donor binding region of MurG, an observation that may be helpful in glycosyltransferase structure prediction. The identification of a conserved structural motif involved in donor binding in different UDP-sugar transferases also suggests that it may be possible to identify—and perhaps alter—the residues that help determine donor specificity.

The lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast-reacting enantiomer in a productive mode and the slow-reacting enantiomer in a nonproductive mode. Nonproductive mode of fast-reacting enantiomers was prohibited by repulsive interactions. For the slow-reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(HNε − Oalc) between the histidine side chain and the alcohol oxygen increases. d(HNε − Oalc) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(HNε − Oalc) is >2.2 Å for slow-reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(HNε − Oalc) is less than 2.0 Å, and slow- and fast-reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E < 100), d(HNε − Oalc) is around 2.1 Å. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols.

We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4-tetrahydro-N-(phenylmethyl)-9-acridinamine and 1,2,3,4-tetrahydro-N-(3-iodophenyl-methyl)-9-acridinamine—all three at 2.7 Å resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active-site gorge, but some of the surface loops deviate by up to 8 Å from their position in the vertebrate structures, and the C-terminal helix is shifted substantially. The active-site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is ∼50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active-site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three-dimensional structures.

The ecdysone receptor (ECR), a nuclear transcription factor controlling insect development, is a novel target for insecticides such as dibenzoylhydrazines with low environmental and toxicological impacts. To understand the high selectivity of such synthetic molecules toward ECR, two homology models of the Chironomus tentans ECR ligand-binding domain (LDB) have been constructed by taking as templates the known LBD crystal structures of the retinoic acid and vitamin D receptors. Docking of 20-hydroxyecdysone (20E) and dibenzoylhydrazines to the receptor suggests a novel superposition of the natural and synthetic molecules; the N-tert-butyl substituent of the dibenzoylhydrazines extends significantly beyond the 20E volume. Our ECR–LBD protein models rationalize how 20E and dibenzoylhydrazines interact with the ligand-binding pocket. The homology model complexes provide new insights that can be exploited in the rational design of new environmentally safe insecticides.

The amino terminal domain of enzyme I (residues 1–258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, ΔH ≅ 140 ± 10 (160) kcal mol−1 and ΔCp ≅ 2.7 (3.3) kcal K−1 mol−1. Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) °C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO32− due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm ∼ 66–68 °C). This possibly arises from an ion pair interaction between the γ-carboxyl of Glu189 and the ε-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K′A = 3 ± 1 × 105 M−1 for EIN and ∼1.2 × 105 M−1 for EI.

Several de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable β-sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N-terminus and positively charged arginine or lysine residues located toward the C-terminus. This arrangement of charge balances the α-helical dipole moment (C → N), resulting in a strong tendency to form stable α-helices as well. Therefore, these peptides can form both stable α-helices and β-sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable β-sheet and α-helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.

We have developed a method for the prediction of an amino acid sequence that is compatible with a three-dimensional backbone structure. Using only a backbone structure of a protein as input, the algorithm is capable of designing sequences that closely resemble natural members of the protein family to which the template structure belongs. In general, the predicted sequences are shown to have multiple sequence profile scores that are dramatically higher than those of random sequences, and sometimes better than some of the natural sequences that make up the superfamily. As anticipated, highly conserved but poorly predicted residues are often those that contribute to the functional rather than structural properties of the protein. Overall, our analysis suggests that statistical profile scores of designed sequences are a novel and valuable figure of merit for assessing and improving protein design algorithms.

Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H-bonds). For this purpose, the solution structure of p16INK4A, whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-mean-square deviation (RMSD) improved from 1.23 to 0.68 Å for backbone heavy atoms. The completion of p15INK4B coupled with refinement of p16INK4A made it possible to compare the structures of the four INK4 members in depth, and to compare the structures of p16INK4A in the free form and in the p16INK4A-CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.

Understanding protein folding requires the determination of the configurational space accessible to the protein at different stages in folding. Here, computer simulation analysis of small angle neutron scattering results is used to probe the change in the distribution of configurations on strong denaturation of a globular protein, phosphoglycerate kinase, in 4 M guanidine hydrochloride solution. To do this atomic-detail ensembles of the unfolded protein chain are modeled and their scattering profiles compared with the experiment. The local conformational statistics are found to strongly influence the experimental intensity at scattering vectors between 0.05 and 0.3 Å−1. Denaturation leads to a reduction in the protein atom-pair distance distribution function over the ∼3–15 Å region that is associated with a quantifiable shift in the backbone torsional angle (φ,ψ) distribution toward the β region of the Ramachandran plot.

The Nef protein of human immunodeficiency virus type I (HIV-1) is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5′-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (≥50 °C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate ≈ 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the α-helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5′-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to ≈50 °C, and (2) stabilizes the covalent protein cofactor linkage up to ≈70 °C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 °C of ≈25 mM. The extra stability conferred by oxyanion–ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a ≈20 °C higher value on the temperature scale.

We describe a new classifier for protein secondary structure prediction that is formed by cascading together different types of classifiers using neural networks and linear discrimination. The new classifier achieves an accuracy of 76.7% (assessed by a rigorous full Jack-knife procedure) on a new nonredundant dataset of 496 nonhomologous sequences (obtained from G.J. Barton and J.A. Cuff). This database was especially designed to train and test protein secondary structure prediction methods, and it uses a more stringent definition of homologous sequence than in previous studies. We show that it is possible to design classifiers that can highly discriminate the three classes (H, E, C) with an accuracy of up to 78% for β-strands, using only a local window and resampling techniques. This indicates that the importance of long-range interactions for the prediction of β-strands has been probably previously overestimated.

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15N NMR-derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross-relaxation rates (ηxy), and steady state {1H}–15N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 °C for 89–100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/ηxy was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter (S2) was used to calculate the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temperature (T*), representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ∼0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ∼9 °C lower (78 °C rather than 87 °C). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as α-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like α-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of HL from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate ∼0.1 s−1, which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.

All the protein sequences from SWISS-PROT database were analyzed for occurrence of single amino acid repeats, tandem oligo-peptide repeats, and periodically conserved amino acids. Single amino acid repeats of glutamine, serine, glutamic acid, glycine, and alanine seem to be tolerated to a considerable extent in many proteins. Tandem oligo-peptide repeats of different types with varying levels of conservation were detected in several proteins and found to be conspicuous, particularly in structural and cell surface proteins. It appears that repeated sequence patterns may be a mechanism that provides regular arrays of spatial and functional groups, useful for structural packing or for one to one interactions with target molecules. To facilitate further explorations, a database of Tandem Repeats in Protein Sequences (TRIPS) has been developed and is available at URL: http://www.ncl-india.org/trips.

The backbone dynamics and overall tumbling of protein G have been investigated using 15N relaxation. Comparison of measured R2/R1 relaxation rate ratios with known three-dimensional coordinates of the protein show that the rotational diffusion tensor is significantly asymmetric, exhibiting a prolate axial symmetry. Extensive Monte Carlo simulations have been used to estimate the uncertainty due to experimental error in the relaxation rates to be D∥/D⊥ = 1.68 ± 0.08, while the dispersion in the NMR ensemble leads to a variation of D∥/D⊥ = 1.65 ± 0.03. Incorporation of this tensorial description into a Lipari–Szabo type analysis of internal motion has allowed us to accurately describe the local dynamics of the molecule. This analysis differs from an earlier study where the overall rotational diffusion was described by a spherical top. In this previous analysis, exchange parameters were fitted to many of the residues in the alpha helix. This was interpreted as reflecting a small motion of the alpha helix with respect to the beta sheet. We propose that the differential relaxation properties of this helix compared to the beta sheet are due to the near-orthogonality of the NH vectors in the two structural motifs with respect to the unique axis of the diffusion tensor. Our analysis shows that when anisotropic rotational diffusion is taken into account NH vectors in these structural motifs appear to be equally rigid. This study underlines the importance of a correct description of the rotational diffusion tensor if internal motion is to be accurately investigated.

Guanine-containing mono- and dinucleotides bind to the active site of ribonuclease A in a nonproductive mode (retro-binding) (Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA, 1992, J Mol Biol 224:265–267). Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro-binding, we determined the structure of two new complexes of RNase A with uridylyl(2′,5′)guanosine obtained by soaking two different forms of pre-grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X-ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 Å (R-factor 0.192) and 2.0 Å (R-factor 0.178), respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro-binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand-bound and a ligand-free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site-directed mutagenesis studies.

Comparison of the inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus to that of Pseudomonas mevalonii HMG-CoA reductase suggested that AF1736 might encode a Class II HMG-CoA reductase. Following polymerase chain reaction–based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85 °C, ΔHa was 54 kJ/mol, and the statin drug mevinolin inhibited competitively with HMG-CoA (Ki 180 μM). Protonated forms of His390 and Lys277, the apparent cognates of the active site histidine and lysine of the P. mevalonii enzyme, appear essential for activity. The mechanism proposed for catalysis of P. mevalonii HMG-CoA reductase thus appears valid for A. fulgidus HMG-CoA reductase. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four reactions revealed that optimal activity using NADP(H) occurred at a pH from 1 to 3 units more acidic than that observed using NAD(H). Kinetic parameters were therefore determined for all substrates for all four catalyzed reactions using either NAD(H) or NADP(H). NADPH and NADH compete for occupancy of a common site. kcat[NAD(H)]/kcat[NADP(H)] varied from unity to under 70 for the four reactions, indicative of slight preference for NAD(H). The results indicate the importance of the protonated status of active site residues His390 and Lys277, shown by altered KM and kcat values, and indicate that NAD(H) and NADP(H) have comparable affinity for the same site.

To investigate the cooperativity of hydrophobic interactions, the potential of mean force of two- and three-molecule methane clusters in water was determined by molecular dynamics simulations using two methods: umbrella-sampling with the weighted histogram analysis method and thermodynamic integration. Two water models, TIP3P and TIP4P, were used, while each methane molecule was modeled as a united atom. It was found that the three-body potential of mean force is not additive, i.e., it cannot be calculated as a sum of two-body contributions, but requires an additional three-body cooperative term. The cooperative term, which amounts to only about 10% of the total hydrophobic association free energy, was found to increase the strength of hydrophobic association; this finding differs from the results of earlier Monte Carlo studies with the free energy perturbation method of Rank and Baker (1997). As in the work of Rank and Baker, the solvent contribution to the potential of mean force was found to be well approximated by the molecular surface of two methane molecules. Moreover, we also found that the cooperative term is well represented by the difference between the molecular surface of the three-methane cluster and those of all three pairs of methane molecules. In addition, it was found that, while there is a cooperative contribution to the hydrophobic association free energy albeit a small one, the errors associated with the use of pairwise potentials are comparable to or larger than this contribution.

The monomer–dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix–helix packing in a membrane-mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C-terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two-stage model for membrane protein folding and oligomerization.