Experimental and theoretical studies of the three-dimensional structure of human interleukin-4

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.     

The structure of rubredoxin from Desulfovibrio desulfuricans strain 27774 at 1.5 A resolution

The structure of a small rubredoxin from the bacterium Desulfovibrio desulfuricans has been determined and refined at 1.5 A resolution. The hairpin loop containing seven residues in other rubredoxins is missing in this 45 residue molecule, and once that fact was determined by amino acid sequencing studies, refinement progressed smoothly to an R value of 0.093 for all reflections from 5 to 1.5 A resolution. Nearly all of the water molecules in the well-ordered triclinic unit cell have been added to the crystallographic model. As in the other refined rubredoxin models, the Fe-S4 complex is slightly distorted from ideal tetrahedral coordination.     

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.     

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.     

Refined structure of bovine carbonic anhydrase III at 2.0 Å resolution

The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.     

Crystal structure of the alpha1beta1 integrin I domain in complex with an antibody Fab fragment

The alpha1beta1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human alpha1beta1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the alpha1beta1 integrin and the Fab fragment of humanized AQC2 antibody. The structure of the complex shows that the antibody blocks the collagen binding site of the I domain. An aspartate residue, from the CDR3 loop of the antibody heavy chain, coordinates the MIDAS metal ion in a manner similar to that of a glutamate residue from collagen. Substitution of the aspartate residue by alanine or arginine results in significant reduction of antibody binding affinity. Interestingly, although the mode of metal ion coordination resembles that of the open conformation, the I domain maintains an overall closed conformation previously observed only for unliganded I domains.     

The three-dimensional structure of glutathione reductase from Escherichia coli at 3.0 A resolution

The structure of glutathione reductase from Escherichia coli has been solved at 3 A resolution using multiple isomorphous replacement, solvent flattening, and molecular replacement on the basis of the homologous (53% identical residues) and structurally well-established human enzyme. The structures of both enzyme species agree with each other in a global way; there is no domain rearrangement. In detail, clear structural differences can be observed. The structure analysis of the E. coli enzyme was tackled in order to understand site-directed mutants, the most spectacular of which changed the cofactor specificity of this enzyme from NADP to NAD (Scrutton et al., 1990, Nature 343:38-43).     

Mutagenesis of histidine 26 demonstrates the importance of loop-loop and loop-protein interactions for the function of iso-1-cytochrome c.

In yeast iso-1-cytochrome c, the side chain of histidine 26 (His26) attaches omega loop A to the main body of the protein by forming a hydrogen bond to the backbone atom carbonyl of glutamic acid 44. The His26 side chain also forms a stabilizing intra-loop interaction through a hydrogen bond to the backbone amide of asparagine 31. To investigate the importance of loop-protein attachment and intra-loop interactions to the structure and function of this protein, a series of site-directed and random-directed mutations were produced at His26. Yeast strains expressing these variant proteins were analyzed for their ability to grow on non-fermentable carbon sources and for their intracellular production of cytochrome c. While the data show that mutations at His26 lead to slightly decreased intracellular amounts of cytochrome c, the level of cytochrome c function is decreased more. The data suggest that cytochrome c reductase binding is affected more than cytochrome c oxidase or lactate dehydrogenase binding. We propose that mutations at this residue increase loop mobility, which, in turn, decreases the protein's ability to bind redox partners.     

Protein structure determination using a database of interatomic distance probabilities

The accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X-ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side-chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side-chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high-resolution refinements were improved, as is shown by an R-factor analysis. The improvements were greatest when refinements were carried out using X-ray data truncated at 3.5 A. The database potential should therefore be a valuable tool for determining X-ray structures, especially when only low-resolution data are available.     

Crystallographic Refinement and Structure-Factor Time-Averaging by Molecular Dynamics in the Absence of a Physical Force Field

Abstract

The application of Molecular-Dynamics simulation in protein-crystallographic structure refinement has become common practice. In this paper, structure optimizations are described where the driving force is derived only from the crystallographic data and not from any physical potential energy function. Under this extreme condition ab initio structure refinement and the application of structure-factor time averaging was investigated using a small 9 atom test system. Success in ab initio refinement, where the starting atomic positions are randomly distributed, depends on the resolution of the crystallographic data used in the optimization. The presence of high resolution data introduces false minima in the X-ray energy profile, enhancing the search problem significantly. On the same system, we also tested the method of time-averaged crystallographically restrained Molecular Dynamics, again in the absence of a physical force field. In this method, the diffraction data is modelled by an ensemble of structures instead of one single structure. In comparison to conventional single-structure refinement, more reflections were required to determine a correct atomic distribution. A time-averaging simulation at 0.2 nm resolution (40 reflections) yielded an incorrect distribution, although a low R-factor was obtained. Simulations at 0.1 nm resolution (248 reflections) gave both low R-factors, 3 to 4%, and correct atomic distributions. The scale factor between the observed and time-averaged calculated structure factor amplitudes appeared to be unstable, when optimized during a time-averaging simulation. Tests of time-averaged restrained simulations with noise added to the observed structure-factor amplitudes, indicated that noise is modelled when no information in the form of constraints or restraints is available to distinguish it from real data.     

人白细胞介素18半胱氨酸的定点突变及其对活性的影响

为研究IL 18结构与功能的关系 ,用重叠延伸PCR定点突变技术构建人白细胞介素 18(hIL 18) 4个半胱氨酸的突变体hIL 18C74 S、C10 4 S、C112 S和C163 S。将突变体的cDNA与原核细胞表达载体pJW2重组并转化大肠杆菌JM10 1。经热诱导后 ,4个突变体在大肠杆菌中均得到了高效表达。表达的蛋白质主要以包涵体的形式存在。包涵体经超声破碎 ,2mol/L尿素洗涤 ,8mol/L尿素溶解 ,SephadexG 10 0柱纯化后 ,纯度可达 90 %以上。以诱导人外周血单个核细胞 (PBMC)产生IFN γ的能力为指标检测复性突变体的活性。结果显示除C10 4 S外 ,其他 3个突变体的生物活性均低于野生型hIL 18,C74 S、C112 S和C163 S的活性分别是野生型hIL 18活性的 5 %、5 8%和11%。证明Cys74 、Cys163 为hIL 18诱导产生IFN γ的功能所必需     

The role of arginine 143 in the electrostatics and mechanism of Cu,Zn superoxide dismutase: Computational and experimental evaluation by mutational analysis

Cu, Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu, Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited pro-tonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu, Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. © 1994 Wiley-Liss, Inc.     

Localized solution structure refinement of an F45W variant of ubiquitin using stochastic boundary molecular dynamics and NMR distance restraints.

The local structure within an 8-A radius around residue 45 of a recombinant F45W variant of human ubiquitin has been determined using 67 interproton distance restraints measured by two-dimensional proton NMR. Proton chemical shift evidence indicates that structural perturbations due to the F45W mutation are minimal and limited to the immediate vicinity of the site of mutation. Simulated annealing implemented with stochastic boundary molecular dynamics was applied to refine the structure of Trp 45 and 10 neighboring residues. The stochastic boundary method allowed the entire protein to be reassembled from the refined coordinates and the outlying unrefined coordinates with little distortion at the boundary. Refinement began with four low-energy indole ring orientations of F45W-substituted wild-type (WT) ubiquitin crystal coordinates. Distance restraints were derived from mostly long-range NOE cross peaks with 51 restraints involving the Trp 45 indole ring. Tandem refinements of 64 structures were done using either (1) upper and lower bounds derived from qualitative inspection of NOE crosspeak intensities or (2) quantitative analysis of cross-peak heights using the program MARDIGRAS. Though similar to those based on qualitative restraint, structures obtained using quantitative NOE analysis were superior in terms of precision and accuracy as measured by back-calculated sixth-root R factors. The six-membered portion of the indole ring is nearly coincident with the phenyl ring of the WT and the indole NH is exposed to solvent. Accommodation of the larger ring is accompanied by small perturbations in the backbone and a 120 degrees rotation of the chi 2 dihedral angle of Leu 50.     

Crystal structure of the cytokine interleukin-1 beta.

The crystal structure of human recombinant interleukin-1 beta has been determined at 3.0 A resolution by the isomorphous replacement method in conjunction with solvent flattening techniques. The model prior to refinement has a crystallographic R-factor of 42.3%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no strong internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by McLachlan (1979) J. Mol. Biol., 133, 557-563, for soybean trypsin inhibitor.     

Hydration of a hydrophobic cavity and its functional role: a simulation study of human interleukin-1beta

Molecular dynamics simulations reveal that the hydrophobic cavity in human cytokine Interleukin-1beta is hydrated and can dynamically accommodate between one and four water molecules. These waters have residence times > 500 ps and can give rise to detectable NOEs, in agreement with NMR observations of Ernst et al. (Science 1995; 267:1813-1817). The waters also display high positional disorder within the cavity, which explains why they have not been resolved crystallographically. The average distribution of water molecules over time within the cavity matches well the low resolution electron density extracted by Yu et al. (Proc Natl Acad Sci 1999; 96:103-108). The water molecules hydrate the hydrophobic cavity preferentially as complex clusters. These clusters result from a combination of hydrogen bonds between the waters and stabilizing interactions between the waters and aromatic rings forming the cavity. Free energy estimates suggest that it takes 4-waters to hydrate the cavity in a thermodynamically stable manner leading to a gain in free energy of transfer from bulk of approximately approximately 3.6 kcal/mol. This arises from the existence of the water clusters in multiple hydrogen bonded states. In addition, the waters are found to migrate either individually or as clusters out of the cavity through several pathways. The upper limit for one-dimensional diffusion of the waters within the protein matrix is 4 A/ps (relative to 6 A/ps for bulk). Simulations reveal pathways in addition to those identified crystallographically, with motions controlled by the rotations of sidechains. We find that only when the hydrophobic cavity is hydrated, do correlated motions couple distant sites with the sites that make contact with the receptor and this data partly offers an explanation of experimental mutagenesis data. Simulations, together with recent observations based on mutagenesis by Heidary et al. (J Mol Biol 2005; 353:1187-1198) that hydrogen bond networks couple motions across long distances in interleukin-1beta, lead us to hypothesize that the hydration of the cavity (conserved across mammals) can thermodynamically enhance hydrogen bond networks to enable coupling across long distances by acting as a plug and this in turn enables a kinetic control of the rate of transmission of signals.     

Structure of the PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions

Tensin is a cytoskeletal protein that links integrins to the actin cytoskeleton at sites of cell-matrix adhesion. Here we describe the crystal structure of the phosphotyrosine-binding (PTB) domain of tensin1, and show that it binds integrins in an NPxY-dependent fashion. Alanine mutagenesis of both the PTB domain and integrin tails supports a model of integrin binding similar to that of the PTB-like domain of talin. However, we also show that phosphorylation of the NPxY tyrosine, which disrupts talin binding, has a negligible effect on tensin binding. This suggests that tyrosine phosphorylation of integrin, which occurs during the maturation of focal adhesions, could act as a switch to promote the migration of tensin-integrin complexes into fibronectin-mediated fibrillar adhesions.     

Salt Bridge Swapping in the EXXERFXYY Motif of Proton-coupled Oligopeptide Transporters

Proton-coupled oligopeptide transporters (POTs) couple the inward transport of di- or tripeptides with an inwardly directed transport of protons. Evidence from several studies of different POTs has pointed toward involvement of a highly conserved sequence motif, E₁XXE₂RFXYY (from here on referred to as E₁XXE₂R), located on Helix I, in interactions with the proton. In this study, we investigated the intracellular substrate accumulation by motif variants with all possible combinations of glutamate residues changed to glutamine and arginine changed to a tyrosine, the latter being a natural variant found in the Escherichia coli POT YjdL. We found that YjdL motif variants with E₁XXE₂R, E₁XXE₂Y, E₁XXQ₂Y, or Q₁XXE₂Y were able to accumulate peptide, whereas those with E₁XXQ₂R, Q₁XXE₂R, or Q₁XXQ₂Y were unable to accumulate peptide, and Q₁XXQ₂R abolished uptake. These results suggest a mechanism that involves swapping of an intramotif salt bridge, i.e. R-E₂ to R-E₁, which is consistent with previous structural studies. Molecular dynamics simulations of the motif variants E₁XXE₂R and E₁XXQ₂R support this mechanism. The simulations showed that upon changing conformation arginine pushes Helix V, through interactions with the highly conserved FYING motif, further away from the central cavity in what could be a stabilization of an inward facing conformation. As E₂ has been suggested to be the primary site for protonation, these novel findings show how protonation may drive conformational changes through interactions of two highly conserved motifs.     

Structural features of the combining site region of Erythrina corallodendron lectin: role of tryptophan 135.

The role of Trp 135 and Tyr 108 in the combining site of Erythrina corallodendron lectin (ECorL) was investigated by physicochemical characterization of mutants obtained by site-directed mutagenesis, hemagglutination-inhibition studies, and molecular modeling, including dynamics simulations. The findings demonstrate that Trp 135 in ECorL: (1) is required for the tight binding of Ca2+ and Mn2+ to the lectin because mutation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) contributes to the high affinity of methyl alpha-N-dansylgalactosaminide (MealphaGalNDns) to the lectin; and (3) is solely responsible for the fluorescence energy transfer between the aromatic residues of the lectin and the dansyl group in the ECorL-MealphaGalNDns complex. Docking of MealphaGalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as required for efficient fluorescence energy transfer, in one of the two possible conformations that this ligand assumes in the bound state. In the W135A mutant, which still binds MealphaGalNDns strongly, the dansyl group may partially insert itself into the place formerly occupied by Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assumes an open conformation. However, a small fraction of the W135A molecules must be able to bind MealphaGalNDns in order to explain the relatively high affinity, as compared to galactose, still remaining for this ligand. A model for the molecular events leading to inactivation of the W135A mutant upon demetallization is also presented in which the cis-trans isomerization of the Ala 88-Asp 89 peptide bond, observed in high-temperature dynamics simulations, appears not to be a required step.     

The native energy landscape for interleukin-1beta. Modulation of the population ensemble through native-state topology

A minimalist Go-model, with no energetic frustration in the native conformation, has been shown to describe accurately the folding pathway of the beta-trefoil protein, interleukin-1beta (IL-1beta). While it appears that these models successfully model transition states and intermediates between the unfolded and native ensembles, it is unclear how accurately they capture smaller, yet biologically relevant, structural changes within the native ensemble after energetic perturbation. Here, we address the following questions. Can a simple Go-model of interleukin-1beta, based on native topology, describe changes in structural properties of the native ensemble as the protein stability is changed? Or is it necessary to include a more explicit representation of atoms, electrostatic, hydrogen bonding, and van der Waals forces to describe these changes? The native ensemble of IL-1beta was characterized using a variety of experimental probes under native (0 M NaCl, guanidine hydrochloride (Gdn-HCl)), moderately destabilized (0 M NaCl, 0.8 M Gdn-HCl), and in moderate salt concentration (0.8 M NaCl, 0 M Gdn-HCl). Heteronuclear (1)H-(15)N nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) NMR spectra confirmed that the beta-trefoil global fold was largely intact under these three conditions. However, 25 of the 153 residues throughout the chain did demonstrate (13)C and (1)H-(15)N chemical shifts when perturbed with 0.8 M NaCl or Gdn-HCl. Despite large differences in protection factors from solvent hydrogen-deuterium exchange for all residues between stable (0 M Gdn-HCl) and destabilized (0.8 M Gdn-HCl) IL-1beta, no difference in steady-state (15)N-(1)H NOE enhancements were measured. Thus, the chemical shifts correlate with a global but limited increase in residue flexibility in the presence of Gdn-HCl. Minimalist simulations highlight the regions of greatest position shift between native and 0.8 M Gdn-HCl, which were determined experimentally. This correlation demonstrates that structural changes within the native ensemble of IL-1beta are, at least partially, governed by the principle of minimal energetic frustration.