Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus

Type IV pili are surface-exposed filaments, which extend from a variety of bacterial pathogens and play a major role in pathogenesis, motility, and DNA uptake. Here, we present the crystal structure of a complex between a cytoplasmic component of the type IV pilus biogenesis system from Thermus thermophilus, PilM, in complex with a peptide derived from the cytoplasmic portion of the inner membrane protein PilN. PilM also binds ATP, and its structure is most similar to the actin-like protein FtsA. PilN binds in a narrow channel between the 1A and 1C subdomains in PilM; the binding site is well conserved in other gram-negative bacteria, notably Neisseria meningitidis, Pseudomonas aeruginosa, and Vibrio cholerae. We find no evidence for the catalysis of ATP hydrolysis by PilM; fluorescence data indicate that the protein is likely to be saturated by ATP at physiological concentrations. In addition, binding of the PilN peptide appears to influence the environment of the ATP binding site. This is the first reported structure of a complex between two type IV pilus biogenesis proteins. We propose a model in which PilM binds ATP and then PilN as one of the first steps in the formation of the inner membrane platform of the type IV pilus biogenesis complex.     

Modeling membrane proteins: The importance of cysteine amino-acids

Computational modeling of membrane proteins is critical to understand biochemical systems and to support chemical biology. In this work, we use a dataset of 448 non-redundant membrane protein chains to expose a “rule” that governs membrane protein structure: free cysteine thiols are not found accessible to oxidative compartments such as the extracellular space, but are rather involved in disulphide bridges. Taking as examples the 1018 three-dimensional models produced during the GPCR Dock, 2008, GPCR Dock, 2010, GPCR Dock, 2013 competitions and 390 models for a GPCR target in CASP13, we show that this rule was not accounted for by the modeling community. We thus highlight a new direction for model development that should lead to more accurate membrane protein models, especially in the loop domains.     

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.     

SAXSDom: Modeling multidomain protein structures using small-angle X-ray scattering data

Many proteins are composed of several domains that pack together into a complex tertiary structure. Multidomain proteins can be challenging for protein structure modeling, particularly those for which templates can be found for individual domains but not for the entire sequence. In such cases, homology modeling can generate high quality models of the domains but not for the orientations between domains. Small-angle X-ray scattering (SAXS) reports the structural properties of entire proteins and has the potential for guiding homology modeling of multidomain proteins. In this article, we describe a novel multidomain protein assembly modeling method, SAXSDom that integrates experimental knowledge from SAXS with probabilistic Input-Output Hidden Markov model to assemble the structures of individual domains together. Four SAXS-based scoring functions were developed and tested, and the method was evaluated on multidomain proteins from two public datasets. Incorporation of SAXS information improved the accuracy of domain assembly for 40 out of 46 critical assessment of protein structure prediction multidomain protein targets and 45 out of 73 multidomain protein targets from the ab initio domain assembly dataset. The results demonstrate that SAXS data can provide useful information to improve the accuracy of domain-domain assembly. The source code and tool packages are available at https://github.com/jianlin-cheng/SAXSDom .     

Modeling of loops in protein structures

Comparative protein structure prediction is limited mostly by the errors in alignment and loop modeling. We describe here a new automated modeling technique that significantly improves the accuracy of loop predictions in protein structures. The positions of all nonhydrogen atoms of the loop are optimized in a fixed environment with respect to a pseudo energy function. The energy is a sum of many spatial restraints that include the bond length, bond angle, and improper dihedral angle terms from the CHARMM-22 force field, statistical preferences for the main-chain and side-chain dihedral angles, and statistical preferences for nonbonded atomic contacts that depend on the two atom types, their distance through space, and separation in sequence. The energy function is optimized with the method of conjugate gradients combined with molecular dynamics and simulated annealing. Typically, the predicted loop conformation corresponds to the lowest energy conformation among 500 independent optimizations. Predictions were made for 40 loops of known structure at each length from 1 to 14 residues. The accuracy of loop predictions is evaluated as a function of thoroughness of conformational sampling, loop length, and structural properties of native loops. When accuracy is measured by local superposition of the model on the native loop, 100, 90, and 30% of 4-, 8-, and 12-residue loop predictions, respectively, had <2 A RMSD error for the mainchain N, C(alpha), C, and O atoms; the average accuracies were 0.59 +/- 0.05, 1.16 +/- 0.10, and 2.61 +/- 0.16 A, respectively. To simulate real comparative modeling problems, the method was also evaluated by predicting loops of known structure in only approximately correct environments with errors typical of comparative modeling without misalignment. When the RMSD distortion of the main-chain stem atoms is 2.5 A, the average loop prediction error increased by 180, 25, and 3% for 4-, 8-, and 12-residue loops, respectively. The accuracy of the lowest energy prediction for a given loop can be estimated from the structural variability among a number of low energy predictions. The relative value of the present method is gauged by (1) comparing it with one of the most successful previously described methods, and (2) describing its accuracy in recent blind predictions of protein structure. Finally, it is shown that the average accuracy of prediction is limited primarily by the accuracy of the energy function rather than by the extent of conformational sampling.     

Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa

In Gram-negative bacteria, most of the sec-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of 12 Xcp proteins (XcpA and XcpP to XcpZ) organized as a multiproteic complex within the envelope. In this study, by a co-purification approach using a His-tagged XcpZ as a bait, XcpY and XcpZ were found associated together to constitute the most stable functional unit so far isolated from the P. aeruginosa secreton. This subcomplex was also found to interact with XcpR and XcpS to form a XcpRSYZ complex which was isolated under native conditions. Another component, XcpP was not found to be associated to the complex but the results suggest that it can transiently interact with the XcpYZ subcomplex in vivo.     

Inferential backbone assignment for sparse data

This paper develops an approach to protein backbone NMR assignment that effectively assigns large proteins while using limited sets of triple-resonance experiments. Our approach handles proteins with large fractions of missing data and many ambiguous pairs of pseudoresidues, and provides a statistical assessment of confidence in global and position-specific assignments. The approach is tested on an extensive set of experimental and synthetic data of up to 723 residues, with match tolerances of up to 0.5 ppm for and resonance types. The tests show that the approach is particularly helpful when data contain experimental noise and require large match tolerances. The keys to the approach are an empirical Bayesian probability model that rigorously accounts for uncertainty in the data at all stages in the analysis, and a hybrid stochastic tree-based search algorithm that effectively explores the large space of possible assignments.     

Bacteriophage HK97 head assembly

Abstract: The head assembly pathway of bacteriophage HK97 shares many features with head assembly pathways determined for other dsDNA phages, and it also provides examples of novel variations on the basic theme. We describe aspects of two specific steps in the assembly pathway, the covalent cross-linking among the assembled head protein subunits and the cleavage of those subunits that takes place earlier in the pathway. Comparisons of head assembly pathways among different phages, as well as comparisons of the organization of the genes that specify those pathways, suggest the range of different solutions phages have found to common assembly problems and give insight into the evolutionary histories of these assembly processes.     

Identification of Chaperonin Particles in Mammalian Brain Cytosol and of T-Complex Polypeptide 1 as One of Their Components

Abstract: An ˜950-kDa heteromeric particle was purified from guinea-pig and rat brain by sucrose gradient fractionation of post-mitochondrial supernatants. Further purification, by affinity chro-matography on ATP-Sepharose and anion exchange FPLC on MonoQ, yielded a particle with typical chaperonin ultrastructure. One of the component polypeptides was recognized by a monoclonal antibody to murine T-complex polypeptide 1. Brain cytosolic chaperonin particles formed a binary complex with unfolded tubulin subunits. The polypeptide compositions of the cytosolic chaperonin particles appeared very similar between brain and testicular tissues of the same animal, but differed subtly between the guineapig and rat.     

Modeling mutations in protein structures

We describe an automated method for the modeling of point mutations in protein structures. The protein is represented by all non-hydrogen atoms. The scoring function consists of several types of physical potential energy terms and homology-derived restraints. The optimization method implements a combination of conjugate gradient minimization and molecular dynamics with simulated annealing. The testing set consists of 717 pairs of known protein structures differing by a single mutation. Twelve variations of the scoring function were tested in three different environments of the mutated residue. The best-performing protocol optimizes all the atoms of the mutated residue, with respect to a scoring function that includes molecular mechanics energy terms for bond distances, angles, dihedral angles, peptide bond planarity, and non-bonded atomic contacts represented by Lennard-Jones potential, dihedral angle restraints derived from the aligned homologous structure, and a statistical potential for non-bonded atomic interactions extracted from a large set of known protein structures. The current method compares favorably with other tested approaches, especially when predicting long and flexible side-chains. In addition to the thoroughness of the conformational search, sampled degrees of freedom, and the scoring function type, the accuracy of the method was also evaluated as a function of the flexibility of the mutated side-chain, the relative volume change of the mutated residue, and its residue type. The results suggest that further improvement is likely to be achieved by concentrating on the improvement of the scoring function, in addition to or instead of increasing the variety of sampled conformations.     

Assembly of protein structure from sparse experimental data: An efficient Monte Carlo model

A new, efficient method for the assembly of protein tertiary structure from known, loosely encoded secondary structure restraints and sparse information about exact side chain contacts is proposed and evaluated. The method is based on a new, very simple method for the reduced modeling of protein structure and dynamics, where the protein is described as a lattice chain connecting side chain centers of mass rather than Cαs. The model has implicit built-in multibody correlations that simulate short- and long-range packing preferences, hydrogen bonding cooperativity and a mean force potential describing hydrophobic interactions. Due to the simplicity of the protein representation and definition of the model force field, the Monte Carlo algorithm is at least an order of magnitude faster than previously published Monte Carlo algorithms for structure assembly. In contrast to existing algorithms, the new method requires a smaller number of tertiary restraints for successful fold assembly; on average, one for every seven residues as compared to one for every four residues. For example, for smaller proteins such as the B domain of protein G, the resulting structures have a coordinate root mean square deviation (cRMSD), which is about 3 Å from the experimental structure; for myoglobin, structures whose backbone cRMSD is 4.3 Å are produced, and for a 247-residue TIM barrel, the cRMSD of the resulting folds is about 6 Å. As would be expected, increasing the number of tertiary restraints improves the accuracy of the assembled structures. The reliability and robustness of the new method should enable its routine application in model building protocols based on various (very sparse) experimentally derived structural restraints. Proteins 32:475–494, 1998. © 1998 Wiley-Liss, Inc.     

Systematic interpolation method predicts protein chromatographic elution from batch isotherm data without a detailed mechanistic isotherm model

Predicting protein elution for overloaded ion exchange columns requires models capable of describing protein binding over broad ranges of protein and salt concentrations. Although approximate mechanistic models are available, they do not always have the accuracy needed for precise predictions. The aim of this work is to develop a method to predict protein chromatographic behavior from batch isotherm data without relying on a mechanistic model. The method uses a systematic empirical interpolation (EI) scheme coupled with a lumped kinetic model with rate parameters determined from HETP measurements for non‐binding conditions, to numerically predict the column behavior. For two experimental systems considered in this work, predictions based on the EI scheme are in excellent agreement with experimental elution profiles under highly overloaded conditions without using any adjustable parameters. A qualitative study of the sensitivity of predicting protein elution profiles to the precision, granularity, and extent of the batch adsorption data shows that the EI scheme is relatively insensitive to the properties of the dataset used, requiring only that the experimental ranges of protein and salt concentrations overlap those under which the protein actually elutes from the column and possess a ±10% measurement precision.     

Molecular modelling of miraculin: Structural analyses and functional hypotheses

Miraculin is a plant protein that displays the peculiar property of modifying taste by swiching sour into a sweet taste. Its monomer is flavourless at all pH as well as at high concentration; the dimer form elicits its taste-modifying activity at acidic pH; a tetrameric form is also reported as active. Two histidine residues, located in exposed regions, are the main responsible of miraculin activity, as demonstrated by mutagenesis studies. Since structural data of miraculin are not available, we have predicted its three-dimensional structure and simulated both its dimer and tetramer forms by comparative modelling and molecular docking techniques. Finally, molecular dynamics simulations at different pH conditions have indicated that at acidic pH the dimer assumes a widely open conformation, in agreement with the hypotheses coming from other studies.     

Myostatin三维结构模建及分子进化分析

Myostatin(MST)为肌肉生长负调节因子,其功能受抑制可导致肌肉量增加.对MST核酸序列进行序列比对,构建进化树;采用同源模建方法首次模建MST成熟肽生物活性二聚体的四级结构,并预测MST与其受体ActRIIB的相互作用模式.进化树将肌肉生长抑制素基因(MSTN)分成4个亚家族:哺乳动物MSTN,鸟类MSTN以及鱼类MSTN 1和2.MST受纯化选择作用,在不同物种的直系同源基因具有较高的刚源性,其中哺乳动物、鸟类MST C端活性肽氨基酸序列高度保守.表明哺乳动物、鸟类MST的结构、功能类似,且信号传导路径可能一致;而鱼类MST的调控机制可能存在较大差异.MST结构及其表面静电势和疏水氨基酸分布表明静电力和疏水相互作用在MST与其受体结合过程中可能起到十分重要的作用.     

Modeling and simulation of biological systems from image data

This essay provides an introduction to the terminology, concepts, methods, and challenges of image‐based modeling in biology. Image‐based modeling and simulation aims at using systematic, quantitative image data to build predictive models of biological systems that can be simulated with a computer. This allows one to disentangle molecular mechanisms from effects of shape and geometry. Questions like “what is the functional role of shape” or “how are biological shapes generated and regulated” can be addressed in the framework of image‐based systems biology. The combination of image quantification, model building, and computer simulation is illustrated here using the example of diffusion in the endoplasmic reticulum.     

Three-stage assembly of the cysteine synthase complex from Escherichia coli

Control of sulfur metabolism in plants and bacteria is linked, in significant measure, to the behavior of the cysteine synthase complex (CSC). The complex is comprised of the two enzymes that catalyze the final steps in cysteine biosynthesis: serine O-acetyltransferase (SAT, EC 2.3.1.30), which produces O-acetyl-L-serine, and O-acetyl-L-serine sulfhydrylase (OASS, EC 2.5.1.47), which converts it to cysteine. SAT (a dimer of homotrimers) binds a maximum of two molecules of OASS (a dimer) in an interaction believed to involve docking of the C terminus from a protomer in an SAT trimer into an OASS active site. This interaction inactivates OASS catalysis and prevents further binding to the trimer; thus, the system exhibits a contact-induced inactivation of half of each biomolecule. To better understand the dynamics and energetics that underlie formation of the CSC, the interactions of OASS and SAT from Escherichia coli were studied at equilibrium and in the pre-steady state. Using an experimental strategy that initiates dissociation of the CSC at different points in the CSC-forming reaction, three stable forms of the complex were identified. Comparison of the binding behaviors of SAT and its C-terminal peptide supports a mechanism in which SAT interacts with OASS in a non-allosteric interaction involving its C terminus. This early docking event appears to fasten the proteins in close proximity and thus prepares the system to engage in a series of subsequent, energetically favorable isomerizations that inactivate OASS and produce the fully isomerized CSC.     

Disulfide bonds convert small heat shock protein Hsp16.3 from a chaperone to a non-chaperone: implications for the evolution of cysteine in molecular chaperones

Molecular chaperones mainly function in assisting newly synthesized polypeptide folding and protect non-native proteins from aggregation, with known structural features such as the ability of spontaneous folding/refolding and high conformational flexibility. In this report, we verified the assumption that the lack of disulfide bonds in molecular chaperones is a prerequisite for such unique structural features. Using small heat shock protein (one sub-class of chaperones) Hsp16.3 as a model system, our results show the following: (1) Cysteine-free Hsp16.3 wild type protein can efficiently exhibit chaperone activity and spontaneously refold/reassemble with high conformational flexibility. (2) Whereas Hsp16.3 G89C mutant with inter-subunit disulfide bonds formed seems to lose the nature of chaperone proteins, i.e., under stress conditions, it neither acts as molecular chaperone nor spontaneously refolds/reassembles. Structural analysis indicated that the mutant exists as an unstable molten globule-like state, which incorrectly exposes hydrophobic surfaces and irreversibly tends to form aggregates that can be suppressed by the other molecular chaperone (alpha-crystallin). By contrast, reduction of disulfide bond in the Hsp16.3 G89C mutant can significantly recover its character as a molecular chaperone. In light of these results, we propose that disulfide bonds could severely disturb the structure/function of molecular chaperones like Hsp16.3. Our results might not only provide insights into understanding the structural basis of chaperone upon binding substrates, but also explain the observation that the occurrence of cysteine in molecular chaperones is much lower than that in other protein families, subsequently being helpful to understand the evolution of protein family.     

用于蛋白质分子设计的环区模建方法

利用距离约束的数据库搜索方法和接触势能分析技术，提出了一种用来模建蛋白质结构环区的分子模建方法。通过对珠蛋白、丝氨酸蛋白酶、钙结合蛋白和溶菌酶中的５０个环区的模建，证明上述方法是可行的。对总区５０个环区的模建表明，８６％的环区可以模建，只有１４％的环区不能模建。研究结果表明这种方法非常适用于蛋白质工程中的环区模建。