Structural context of exons in protein domains: implications for protein modelling and design

Intron boundaries were extracted from genomic data and mapped onto single-domain human and murine protein structures taken from the Protein Data Bank. A first analysis of this set of proteins shows that intron boundaries prefer to be in non-regular secondary structure elements, while avoiding alpha-helices and beta-strands. This fact alone suggests an evolutionary model in which introns are constrained by protein structure, particularly by tertiary structure contacts. In addition, in silico recombination experiments of a subset of these proteins together with their homologues, including those in different species, show that introns have a tendency to occur away from artificial crossover hot spots. Altogether, these findings support a model in which genes can preferentially harbour introns in less constrained regions of the protein fold they code for. In the light of these findings, we discuss some implications for protein modelling and design.     

Structural and functional conservation profiles of novel cathepsin L-like proteins identified in the Drosophila melanogaster genome

Cathepsin L is a cysteine protease which degrades connective tissue proteins including collagen, elastin, and fibronectin. In this study, five well-characterized cathepsin L proteins from different arthropods were used as query sequences for the Drosophila genome database. The search yielded 10 cathepsin L-like sequences, of which eight putatively represent novel cathepsin L-like proteins. To understand the phylogenetic relationship among these cathepsin L-like proteins, a phylogenetic tree was constructed based on their sequences. In addition, models of the tertiary structures of cathepsin L were constructed using homology modeling methods and subjected to molecular dynamics simulations to obtain reasonable structure to understand its dynamical behavior. Our findings demonstrate that all of the potential Drosophila cathepsin L-like proteins contain at least one cathepsin propeptide inhibitor domain. Multiple sequence alignment and homology models clearly highlight the conservation of active site residues, disulfide bonds, and amino acid residues critical for inhibitor binding. Furthermore, comparative modeling indicates that the sequence/structure/function profiles and active site architectures are conserved.     

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.     

Homology modeling of histidine-containing phosphocarrier protein and eosinophil-derived neurotoxin: Construction of models and comparison with experiment

Homology modeling methods have been used to construct models of two proteins—the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions—possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled—reasons for this and possible enhancements are discussed. © 1995 Wiley-Liss, Inc.     

Structural modeling of protein complexes: Current capabilities and challenges

Proteins frequently interact with each other, and the knowledge of structures of the corresponding protein complexes is necessary to understand how they function. Computational methods are increasingly used to provide structural models of protein complexes. Not surprisingly, community-wide Critical Assessment of protein Structure Prediction (CASP) experiments have recently started monitoring the progress in this research area. We participated in CASP13 with the aim to evaluate our current capabilities in modeling of protein complexes and to gain a better understanding of factors that exert the largest impact on these capabilities. To model protein complexes in CASP13, we applied template-based modeling, free docking and hybrid techniques that enabled us to generate models of the topmost quality for 27 of 42 multimers. If templates for protein complexes could be identified, we modeled the structures with reasonable accuracy by straightforward homology modeling. If only partial templates were available, it was nevertheless possible to predict the interaction interfaces correctly or to generate acceptable models for protein complexes by combining template-based modeling with docking. If no templates were available, we used rigid-body docking with limited success. However, in some free docking models, despite the incorrect subunit orientation and missed interface contacts, the approximate location of protein binding sites was identified correctly. Apparently, our overall performance in docking was limited by the quality of monomer models and by the imperfection of scoring methods. The impact of human intervention on our results in modeling of protein complexes was significant indicating the need for improvements of automatic methods.     

Diverse role of conformational dynamics in carboxypeptidase A-driven peptide and ester hydrolyses: disclosing the "perfect induced fit" and "protein local unfolding" pathways by altering protein stability

Catalytic mechanisms of carboxypeptidase A (CPA) are well known for their diversity and the relative inaccessibility for a decisive comprehension. Recent encouraging attempts through modern computational techniques promoted new challenges for the complementary experimental endeavors. In this work, we have applied the stopped-flow technique and the method of reaction progress curve fitting to extract kinetic parameters for the CPA-catalyzed hydrolyses of smaller (typical) peptide and ester substrates, known for their strong activating/inhibiting impact, thus to which the traditional method of "initial rates" is not applicable. Our approach that innately implies the overall constancy of the affecter (substrate plus "active" product) concentration, made it possible to rigorously determine the physically meaningful "effective" values for the catalytic and Michaelis constants under diverse experimental conditions including variable temperature and urea or trimethylamine N-oxide concentrations. Analysis of the obtained results allowed for: (i) the further substantiation of diverse mechanistic patterns for archetypal specific peptide and ester substrates, (ii) testing and disclosure of intrinsic links between the stabilizing/destabilizing and activating/inhibiting effects for the important model enzyme, CPA, and (iii) tentative explanation of a distinct activating/inhibiting impact of these substrates through the strong specific interaction of their benzyl (Bz) moiety with the substrate binding S(3) subsite of CPA. We have demonstrated that stabilization of CPA either through the interaction with an extra Bz moiety (belonging to another substrate or to the product) leads to the increase of its catalytic power with respect to the specific peptide substrate and to its decrease with respect to the counterpart ester substrate. We conjecture that the catalytic mechanisms operating in these two cases include: (a) the "promoted water" mechanism for the peptide substrate that, seemingly, provides the almost "perfect induced fit" (low-barrier conformational adaptation), and (b) presumably, the "anhydride intermediate" mechanism for the ester substrate that, anyway, requires substantial conformational rearrangement (in fact, "partial or local unfolding") of the protein environment in the course of the rate-determining step.     

In silico structural homology modeling of nif A protein of rhizobial strains in selective legume plants

Symbiosis is a complex genetic regulatory biological evolution which is highly specific pertaining to plant species and microbial strains. Biological nitrogen fixation in legumes is a functional combination of nodulation by nod genes and regulation by nif, fix genes. Three rhizobial strains (Rhizobium leguminosarum, Bradyrhizobium japonicum, and Mesorhizobium ciceri) that we considered for in silico analysis of nif A are proved to be the best isolates with respect to N₂ fixing for ground nut, chick pea and soya bean (in vitro) out of 47 forest soil samples. An attempt has been made to understand the structural characteristics and variations of nif genes that may reveal the factors influencing the nitrogen fixation. The primary, secondary and tertiary structure of nif A protein was analyzed by using multiple bioinformatics tools such as chou-Fasman, GOR, ExPasy ProtParam tools, Prosa -web. Literature shows that the homology modeling of nif A protein have not been explored yet which insisted the immediate development for better understanding of nif A structure and its influence on biological nitrogen fixation. In the present predicted 3D structure, the nif A protein was analyzed by three different software tools (Phyre2, Swiss model, Modeller) and validated accordingly which can be considered as an acceptable model. However further in silico studies are suggested to determine the specific factors responsible for nitrogen fixing in the present three rhizobial strains.     

Generalized comparative modeling (GENECOMP): a combination of sequence comparison, threading, and lattice modeling for protein structure prediction and refinement

An improved generalized comparative modeling method, GENECOMP, for the refinement of threading models is developed and validated on the Fischer database of 68 probe-template pairs, a standard benchmark used to evaluate threading approaches. The basic idea is to perform ab initio folding using a lattice protein model, SICHO, near the template provided by the new threading algorithm PROSPECTOR. PROSPECTOR also provides predicted contacts and secondary structure for the template-aligned regions, and possibly for the unaligned regions by garnering additional information from other top-scoring threaded structures. Since the lowest-energy structure generated by the simulations is not necessarily the best structure, we employed two structure-selection protocols: distance geometry and clustering. In general, clustering is found to generate somewhat better quality structures in 38 of 68 cases. When applied to the Fischer database, the protocol does no harm and in a significant number of cases improves upon the initial threading model, sometimes dramatically. The procedure is readily automated and can be implemented on a genomic scale.     

Structural aspects of the functional modules in human protein kinase-Cα deduced from comparative analyses

Three-dimensional models of the five functional modules in human protein kinase Cα (PKCα) have been generated on the basis of known related structures. The catalytic region at the C-terminus of the sequence and the N-terminal auto-inhibitory pseudo-substrate have been modeled using the crystal structure complex of cAMP-dependent protein kinese (cAPK) and PKI peptide. While the N-terminal helix of the catalytic region of PKCα is predicted to be in a different location compared with cAPK, the C-terminal extension is modeled like that in the cAPK. The predicted permissive phosphorylation site of PKCα, Thr 497, is found to be entirely consistent with the mutagenesis studies. Basic Lys and Arg residues in the pseudo-substrate make several specific interactions with acidic residues in the catalytic region and may interact with the permissive phosphorylation site. Models of the two zinc-binding modules of PKCα are based on nuclear magnetic resonance and crystal structures of such modules in other PKC isoforms while the calcium phospholipid binding module (C2) is based on the crystal structure of a repeating unit in synaptotagmin I. Phorbol ester binding regions in zinc-binding modules and the calcium binding region in the C2 domain are similar to those in the basis structures. A hypothetical model of the relative positions of all five modules has the putative lipid binding ends of the C2 and the two zinc-binding domains pointing in the same direction and may serve as a basis for further experiments. © 1996 Wiley-Liss, Inc.     

Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients

Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype.     

Structural and functional defects caused by point mutations in the alpha-crystallin domain of a bacterial alpha-heat shock protein

The diverse family of alpha-crystallin-type small heat shock proteins (alpha-Hsps or sHsps) is characterised by a central, moderately conserved alpha-crystallin domain. Oligomerisation followed by dissociation of subparticles is thought to be a prerequisite for chaperone function. We demonstrate that HspH, a bacterial alpha-Hsp from the soybean-symbiont Bradyrhizobium japonicum, assembles into dynamic complexes freely exchanging subunits with homologous and heterologous complexes. The importance of the alpha-crystallin domain for oligomerisation and chaperone activity was tested by site-directed mutagenesis of 12 different residues. In contrast to mammalian alpha-Hsps, the majority of these mutations elicited severe structural and functional defects in HspH. The individual exchange of five amino acid residues throughout the alpha-crystallin domain was found to compromise oligomerisation to various degrees. Assembly defects resulting in complexes of reduced size correlated with greatly decreased or abolished chaperone activity, reinforcing that complete oligomerisation is required for functionality. Mutation of a highly conserved glycine (G114) at the C-terminal end of the alpha-crystallin domain specifically impaired chaperone activity without interfering with oligomerisation properties, indicating that this residue is critical for substrate interaction. The structural and functional importance of this and other residues is discussed in the context of a modeled three-dimensional structure of HspH.     

Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg²⁺, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.     

Multiple mapping method: a novel approach to the sequence-to-structure alignment problem in comparative protein structure modeling

A major bottleneck in comparative protein structure modeling is the quality of input alignment between the target sequence and the template structure. A number of alignment methods are available, but none of these techniques produce consistently good solutions for all cases. Alignments produced by alternative methods may be superior in certain segments but inferior in others when compared to each other; therefore, an accurate solution often requires an optimal combination of them. To address this problem, we have developed a new approach, Multiple Mapping Method (MMM). The algorithm first identifies the alternatively aligned regions from a set of input alignments. These alternatively aligned segments are scored using a composite scoring function, which determines their fitness within the structural environment of the template. The best scoring regions from a set of alternative segments are combined with the core part of the alignments to produce the final MMM alignment. The algorithm was tested on a dataset of 1400 protein pairs using 11 combinations of two to four alignment methods. In all cases MMM showed statistically significant improvement by reducing alignment errors in the range of 3 to 17%. MMM also compared favorably over two alignment meta-servers. The algorithm is computationally efficient; therefore, it is a suitable tool for genome scale modeling studies.     

Internal organization of large protein families: relationship between the sequence, structure, and function-based clustering

The protein universe can be organized in families that group proteins sharing common ancestry. Such families display variable levels of structural and functional divergence, from homogenous families, where all members have the same function and very similar structure, to very divergent families, where large variations in function and structure are observed. For practical purposes of structure and function prediction, it would be beneficial to identify sub-groups of proteins with highly similar structures (iso-structural) and/or functions (iso-functional) within divergent protein families. We compared three algorithms in their ability to cluster large protein families and discuss whether any of these methods could reliably identify such iso-structural or iso-functional groups. We show that clustering using profile-sequence and profile-profile comparison methods closely reproduces clusters based on similarities between 3D structures or clusters of proteins with similar biological functions. In contrast, the still commonly used sequence-based methods with fixed thresholds result in vast overestimates of structural and functional diversity in protein families. As a result, these methods also overestimate the number of protein structures that have to be determined to fully characterize structural space of such families. The fact that one can build reliable models based on apparently distantly related templates is crucial for extracting maximal amount of information from new sequencing projects.     

Structural modeling of SoxF protein from Chlorobium tepidum: an approach to understand the molecular basis of thiosulfate oxidation

Microbial redox reactions of inorganic sulfur compounds play a vital role in balancing the turnover of this element in the environment. These vital reactions are carried out by the enzyme system encoded by the sox operon. The central player of the sulfur oxidation biochemistry is the SoxY–Z protein complex. Another protein called SoxF having sulfide dehydrogenase activity has the ability to reactivate the inactivated SoxY–Z protein complex. This SoxF protein is obtained from the sox operon of Chlorobium tepidium. In the present work an attempt has been made to understand the structural details of the activity of SoxF protein. A plausible biochemical mechanism has been predicted regarding the involvement of the SoxF protein in biological sulfur anion oxidation process. Since this is the first report regarding the structural biology of SoxF protein this study may shed light in the hitherto unknown molecular biochemistry of sulfur anion oxidation by sox operon.     

Tumor suppressor INK4: refinement of p16INK4A structure and determination of p15INK4B structure by comparative modeling and NMR data

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 pl6INK4A, 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 A 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.     

Structural mechanisms of constitutive activation in the C5a receptors with mutations in the extracellular loops: molecular modeling study

Previously we demonstrated by random saturation mutagenesis a set of mutations in the extracellular (EC) loops that constitutively activate the C5a receptor (C5aR) (Klco et al., Nat Struct Mol Biol 2005;12:320-326; Klco et al., J Biol Chem 2006;281:12010-12019). In this study, molecular modeling revealed possible conformations for the extracellular loops of the C5a receptors with mutations in the EC2 loop or in the EC3 loop. Comparison of low-energy conformations of the EC loops defined two distinct clusters of conformations typical either for strongly constitutively active mutants of C5aR (the CAM cluster) or for nonconstitutively active mutants (the non-CAM cluster). In the CAM cluster, the EC3 loop was turned towards the transmembrane (TM) helical bundle and more closely interacted with EC2 than in the non-CAM cluster. This suggested a structural mechanism of constitutive activity where EC3 contacts EC2 leading to EC2 interactions with helix TM3, thus triggering movement of TM7 towards TM2 and TM3. The movement initiates rearrangement of the system of hydrogen bonds between TM2, TM3 and TM7 including formation of the hydrogen bond between the side chains of D82(2.50) in TM2 and N296(7.49) in TM7, which is crucial for formation of the activated states of the C5a receptors (Nikiforovich et al., Proteins: Struct Funct Gene 2011;79:787-802). Since the relative large length of EC3 in C5aR (13 residues) is comparable with those in many other members of rhodopsin family of GPCRs (13-19 residues), our findings might reflect general mechanisms of receptor constitutive activation. The very recent X-ray structure of the agonist-induced constitutively active mutant of rhodopsin (Standfuss et al., Nature 2011;471:656-660) is discussed in view of our modeling results.     

Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations

Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.