Structural and functional conservation of Mycobacterium tuberculosis GroEL paralogs suggests that GroEL1 Is a chaperonin

GroEL is a group I chaperonin that facilitates protein folding and prevents protein aggregation in the bacterial cytosol. Mycobacteria are unusual in encoding two or more copies of GroEL in their genome. While GroEL2 is essential for viability and likely functions as the general housekeeping chaperonin, GroEL1 is dispensable, but its structure and function remain unclear.Here, we present the 2.2-Å resolution crystal structure of a 23-kDa fragment of Mycobacterium tuberculosis GroEL1 consisting of an extended apical domain. Our X-ray structure of the GroEL1 apical domain closely resembles those of Escherichia coli GroEL and M. tuberculosis GroEL2, thus highlighting the remarkable structural conservation of bacterial chaperonins. Notably, in our structure, the proposed substrate-binding site of GroEL1 interacts with the N-terminal region of a symmetry-related neighboring GroEL1 molecule. The latter is consistent with the known GroEL apical domain function in substrate binding and is supported by results obtained from using peptide array technology. Taken together, these data show that the apical domains of M. tuberculosis GroEL paralogs are conserved in three-dimensional structure, suggesting that GroEL1, like GroEL2, is a chaperonin.     

GroEL Ring Separation and Exchange in the Chaperonin Reaction

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结核分枝杆菌热休克蛋白GroEL1的研究进展

结核分枝杆菌(Mycobacterium tuberculosis)的GroEL1蛋白是基因复制产物,作为热休克蛋白(HSP)一员,不但行使原有的助折叠分子伴侣功能,而且可作为抗原,在抗原呈递中起作用。另外,由于其独特的结构特点,GroEL1还具有多种生物功能,如作为蛋白分子伴侣,控制细胞因子依赖性肉芽肿的产生;作为DNA分子伴侣,保护DNA免受损害。而在分枝杆菌属的其他物种如耻垢分枝杆菌(Mycobacterium smegmatis)中却行使不同的生物学功能。这些功能产生的原因和机制有待进一步研究,从而为结核病的发生及结核分枝杆菌的进化机制提供更多的依据。     

Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.     

Structural annotation of Mycobacterium tuberculosis proteome

Of the ～4000 ORFs identified through the genome sequence of Mycobacterium tuberculosis (TB) H37Rv, experimentally determined structures are available for 312. Since knowledge of protein structures is essential to obtain a high-resolution understanding of the underlying biology, we seek to obtain a structural annotation for the genome, using computational methods. Structural models were obtained and validated for ～2877 ORFs, covering ～70% of the genome. Functional annotation of each protein was based on fold-based functional assignments and a novel binding site based ligand association. New algorithms for binding site detection and genome scale binding site comparison at the structural level, recently reported from the laboratory, were utilized. Besides these, the annotation covers detection of various sequence and sub-structural motifs and quaternary structure predictions based on the corresponding templates. The study provides an opportunity to obtain a global perspective of the fold distribution in the genome. The annotation indicates that cellular metabolism can be achieved with only 219 folds. New insights about the folds that predominate in the genome, as well as the fold-combinations that make up multi-domain proteins are also obtained. 1728 binding pockets have been associated with ligands through binding site identification and sub-structure similarity analyses. The resource (http://proline.physics.iisc.ernet.in/Tbstructuralannotation), being one of the first to be based on structure-derived functional annotations at a genome scale, is expected to be useful for better understanding of TB and for application in drug discovery. The reported annotation pipeline is fairly generic and can be applied to other genomes as well.     

Acetylome Analysis Reveals Diverse Functions of Lysine Acetylation in Mycobacterium tuberculosis

The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD⁺-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.Mycobacterium tuberculosis was responsible for 1.3 million deaths and 8.6 million new cases of tuberculosis (TB)¹ worldwide in 2012 (1). This global public health crisis remains a serious problem, with the emergence of drug-resistant M. tuberculosis, especially multidrug-resistant and extensively drug-resistant M. tuberculosis, and also the emergence of coinfections of TB and human immunodeficiency virus (2, 3). To counter the increasing threat of TB, it is critical to understand fundamental aspects of TB-related biology. Such studies will not only provide new drug targets for the design of novel therapeutic agents, but also facilitate the development of novel diagnostic tools and new vaccines.Acetylation is one of the important protein modifications and occurs both co- and post-translationally on the α-amino group at the N terminus of the protein, so-called “N-terminal acetylation,” or on the ε-amino group on the side chain of lysine (4). Lysine acetylation is one of the most common post-translational modifications to proteins in both eukaryotes and prokaryotes. As a dynamic and reversible process, protein acetylation plays important roles in many cellular physiological processes, including cell-cycle regulation and apoptosis, cell morphology (5), metabolic pathways (6–8), protein interactions (9), and enzymatic activity (8, 10). In recent years, great advances have been made in proteomic studies, and a large number of lysine-acetylated proteins have been identified in many eukaryotes, including human (5, 11, 12), rat (13), mouse (11), Drosophila (14), Arabidopsis (15, 16), Saccharomyces cerevisiae (17), and protozoans (18, 19). The global analysis of lysine acetylation has also been reported in bacteria, including Escherichia coli (20–22), Erwinia amylovora (23), Bacillus subtilis (24), and Salmonella enterica (6). These acetylome studies have generated large datasets of bacterial proteins acetylated on lysine residues and have demonstrated the diverse cellular functions of lysine acetylation in bacteria.Increasing evidence shows that protein acetylation occurs and plays an important regulatory role in mycobacteria (8, 25–31). For example, Lange et al. reported the N-terminal acetylation of early secreted antigenic target 6 (ESAT-6) protein (31). Rv1151c is reported to be an NAD⁺-dependent protein deacetylase in M. tuberculosis that deacetylates and thus regulates the activity of acetyl-CoA synthase (25, 32). Two cyclic adenosine monophosphate (cAMP)-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) show similarity to the GNAT family of acetyltransferases and could acetylate a universal stress protein (USP, MSMEG_4207) (30). Subsequent structural studies revealed the fine mechanisms of how cAMP regulates the protein lysine acetyltransferase in mycobacteria (27, 28). Very recently, reversible lysine acetylation was shown to regulate the activity of several fatty acyl-CoA synthetases in M. tuberculosis (8, 26), and also to regulate acetate and propionate metabolism in M. smegmatis (8, 26). However, to the best of our knowledge, only a few acetylated proteins in M. tuberculosis have been identified, presenting a major obstacle to further understanding the regulatory roles of reversible lysine acetylation in this life-threatening pathogen.To fill this gap in our knowledge, we undertook a systematic study of the functional roles of lysine acetylation in M. tuberculosis. We performed an acetylomic analysis of M. tuberculosis H37Ra using high-accuracy MS combined with the identification of 226 unique lysine acetylation sites on 137 proteins. This set of M. tuberculosis proteins acetylated on lysine residues supports the emerging view that lysine acetylation is a general and fundamental regulatory process, and is not restricted to eukaryotes. It also opens the way for its detailed functional and evolutionary analysis of lysine acetylation in M. tuberculosis. The identified acetylated proteins that are involved in several important biological processes were functionally categorized into an interaction map. This is the first time that an interaction network of acetylated proteins in M. tuberculosis has been constructed, and should allow us to better understand the significance of acetylation in key cellular mechanisms in M. tuberculosis. To further explore the effects of lysine acetylation on the physiology of M. tuberculosis H37Ra, MRA_1161, the gene encoding the only known protein deacetylase in this bacterium, was deleted. The roles of MRA_1161 in the colony morphology, carbon source utilization, heat stress tolerance, and biofilm formation of M. tuberculosis were analyzed. The effect of lysine acetylation on the immunogenicity of a known immunogen, HspX, was also tested.     

A novel nucleoid-associated protein of Mycobacterium tuberculosis is a sequence homolog of GroEL

The Mycobacterium tuberculosis genome sequence reveals remarkable absence of many nucleoid-associated proteins (NAPs), such as HNS, Hfq or DPS. In order to characterize the nucleoids of M. tuberculosis, we have attempted to identify NAPs, and report an interesting finding that a chaperonin-homolog, GroEL1, is nucleoid associated. We report that M. tuberculosis GroEL1 binds DNA with low specificity but high affinity, suggesting that it might have naturally evolved to bind DNA. We are able to demonstrate that GroEL1 can effectively function as a DNA-protecting agent against DNase I or hydroxyl-radicals. Moreover, Atomic Force Microscopic studies reveal that GroEL1 can condense a large DNA into a compact structure. We also provide in vivo evidences that include presence of GroEL1 in purified nucleoids, in vivo crosslinking followed by Southern hybridizations and immunofluorescence imaging in M. tuberculosis confirming that GroEL1: DNA interactions occur in natural biological settings. These findings therefore reveal that M. tuberculosis GroEL1 has evolved to be associated with nucleoids.     

Levels of Buchnera aphidicola Chaperonin GroEL During Growth of the Aphid Schizaphis graminum

Buchnera aphidicola is the prokaryotic, intracellular symbiont found in the aphid Schizaphis graminum. Using an immunological approach, we have quantitated the amount of the B. aphidicola chaperonin, GroEL, present in aphid cell-free extracts during the growth cycle of S. graminum at 23°C. Our results indicate that the increase in GroEL approximately follows the increase in aphid weight and endosymbiont number for the first 12 days after birth of the aphid. A 9-day-old aphid contains 1.6 × 10⁵ molecules of GroEL per μm³ of cell volume. This number is similar to that found in Escherichia coli growing at 46°C, close to its maximal growth temperature, and a condition at which there is a major increase in the levels of chaperonins and other stress proteins. It is estimated that at 23°C, 10% of the B. aphidicola protein is GroEL. When S. graminum grown at 23°C was shifted to 33°C for 1 day and subsequently to 23°C, there was no change in the level of GroEL or the rate of growth. It is possible that the high level of GroEL in the endosymbiont masked an increase in the protein owing to a heat shock response.     

Chaperonin GroEL meets the substrate protein as a "load" of the rings

Chaperonin GroEL is an essential molecular chaperone that assists protein folding in the cell. With the aid of cochaperonin GroES and ATP, double ring-shaped GroEL encapsulates non-native substrate proteins inside the cavity of the GroEL-ES complex. Although extensive studies have revealed the outline of GroEL mechanism over the past decade, central questions remain: What are the in vivo substrate proteins? How does GroEL encapsulate the substrates inside the cavity in spite of an apparent entropic difficulty? Is the folding inside the GroEL-ES cavity the same as bulk spontaneous folding? In this review I summarize the recent progress on in vivo and in vitro aspects of GroEL. In particular, emerging evidence shows that the substrate protein itself influences the chaperonin GroEL structure and reaction cycle. Finally I propose the mechanistic similarity between GroEL and kinesin, a molecular motor that moves along a microtubule in an ATP-dependent manner.     

Contact Order Is a Determinant for the Dependence of GFP Folding on the Chaperonin GroEL

The GroE chaperonin system facilitates protein folding in an ATP-dependent manner. It has remained unclear why some proteins are obligate clients of the GroE system, whereas other closely related proteins are able to fold efficiently in its absence. Factors that cause folding to be slower affect kinetic partitioning between spontaneous folding and chaperone binding in favor of the latter. One such potential factor is contact order (CO), which is the average separation in sequence between residues that are in contact in the native structure. Here, we generated variants of enhanced green fluorescent protein with different COs using circular permutations. We found that GroE dependence in vitro and in vivo increases with increasing CO. Thus, our results show that CO is relevant not only for folding in vitro of relatively simple model systems but also for chaperonin dependence and folding in vivo.     

Action of the Chaperonin GroEL/ES on a Non-native Substrate Observed with Single-Molecule FRET

The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.     

Designing a High Throughput Refolding Array Using a Combination of the GroEL Chaperonin and Osmolytes

Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL–GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin–substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a ‘folding array’ to identify superior folding conditions. The stable chaperonin–substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (~mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.     

Redundant Function of cmaA2 and mmaA2 in Mycobacterium tuberculosis cis Cyclopropanation of Oxygenated Mycolates

The Mycobacterium tuberculosis cell envelope contains a wide variety of lipids and glycolipids, including mycolic acids, long-chain branched fatty acids that are decorated by cyclopropane rings. Genetic analysis of the mycolate methyltransferase family has been a powerful approach to assign functions to each of these enzymes but has failed to reveal the origin of cis cyclopropanation of the oxygenated mycolates. Here we examine potential redundancy between mycolic acid methyltransferases by generating and analyzing M. tuberculosis strains lacking mmaA2 and cmaA2, mmaA2 and cmaA1, or mmaA1 alone. M. tuberculosis lacking both cmaA2 and mmaA2 cannot cis cyclopropanate methoxymycolates or ketomycolates, phenotypes not shared by the mmaA2 and cmaA2 single mutants. In contrast, a combined loss of cmaA1 and mmaA2 had no effect on mycolic acid modification compared to results with a loss of mmaA2 alone. Deletion of mmaA1 from M. tuberculosis abolishes trans cyclopropanation without accumulation of trans-unsaturated oxygenated mycolates, placing MmaA1 in the biosynthetic pathway for trans-cyclopropanated oxygenated mycolates before CmaA2. These results define new functions for the mycolic acid methyltransferases of M. tuberculosis and indicate a substantial redundancy of function for MmaA2 and CmaA2, the latter of which can function as both a cis and trans cyclopropane synthase for the oxygenated mycolates.Mycobacterium tuberculosis infection is an ongoing global health crisis. Alleviation of this crisis will require a multidisciplinary approach that must include new antibiotics active against M. tuberculosis. A growing body of literature implicates cell envelope lipids in the pathogenesis of M. tuberculosis infection (5-10, 14-15, 20). The enzymatic pathways that synthesize M. tuberculosis cell envelope lipids are the target of presently available antituberculosis antimicrobials and may be candidates for future antibiotic development.The mycolic acids of M. tuberculosis are alpha-alkyl, beta-hydroxy fatty acids which are 75 to 85 carbons in length (3). There are three classes of major mycolic acids: alpha-, methoxy-, and ketomycolates (Fig. ​(Fig.1).1). Whereas all mycobacteria synthesize mycolic acids, only pathogenic mycobacteria (for example, M. tuberculosis, M. leprae, M. avium, and M. bovis) produce significant quantities of mycolic acids with cyclopropane rings, three-member carbon rings which are added to the meromycolate chain (3). Alpha-mycolates have two cis cyclopropane rings, while methoxy- and ketomycolates have either a cis or trans cyclopropane ring at the proximal position, the latter with a distal methyl branch (Fig. ​(Fig.1).1). In contrast to the case with Escherichia coli, which encodes a single cyclopropane fatty acid synthase (CFAS) (16-17), the M. tuberculosis genome encodes a family of S-adenosyl methionine-dependent methyltransferases that modify cell envelope mycolic acids with methyl branches and cyclopropane rings. Despite substantial amino acid identity, systematic characterization of M. tuberculosis null mutants in each of these methyltransferases has revealed highly specific functions which were not revealed when the enzymes were overexpressed in M. smegmatis (12, 26, 28). Deletion of pcaA greatly reduces synthesis of the proximal cyclopropane ring of the alpha-mycolates (15), whereas deletion of mmaA2 greatly reduces the distal cyclopropane of the same lipid (13). Loss of mmaA2 also causes a mild impairment of methoxymycolate, but not ketomycolate, cis cyclopropanation (13). Similar genetic approaches established cmaA2 as the only trans cyclopropane synthase of oxygenated mycolates (14), while loss of mmaA3 abolishes methoxymycolates, a spontaneous mutation found in many M. bovis BCG strains (4, 11). Finally, deletion of mmaA4 abolishes synthesis of both methoxy- and ketomycolates (10). Recent chemical-genetic analysis of this enzyme family indicates that combined inhibition of their function is lethal to M. tuberculosis, strongly supporting an approach targeting this enzyme family for antimicrobial development (2, 8-19, 25).Open in a separate windowFIG. 1.Chemical structures of the major mycolic acids of M. tuberculosis. Cyclopropane rings and methyl branches are shown and annotated with the methyltransferase responsible for their synthesis.In addition to this essential role in combination, recent evidence implicates individual cyclopropane modifications as important determinants of M. tuberculosis host-pathogen interactions. Inactivation of pcaA causes attenuation of M. tuberculosis in the mouse model of infection while stimulating less-severe granulomatous pathology (15, 23). In contrast, deletion of cmaA2 has no effect on bacterial loads during mouse infection but causes hypervirulence while inducing more-severe granulomatous pathology (24). Inactivation of mmaA4, which leads to an absence of methoxy- and ketomycolates, causes a severe growth defect during the first 3 weeks of infection (10). All of these studies implicate the fine structure of mycolic acids in the pathogenesis of M. tuberculosis infection. One mechanism by which cyclopropanation mediates pathogenesis is through altered inflammatory activity of trehalose dimycolate (TDM), an inflammatory glycolipid. The cyclopropane content of TDM is a major determinant of its inflammatory activity, and this altered TDM is responsible for the virulence phenotypes of cyclopropane-deficient M. tuberculosis strains (9, 23-24).Despite major advances in our understanding of the biosynthesis and pathogenetic function of cyclopropanated mycolic acids through genetic approaches, the methyltransferase(s) that synthesizes the cis cyclopropane ring on the methoxy- and ketomycolates is unknown. In addition, the function of the MmaA1 methyltransferase has not been explored through construction of a null mutant. Prior experiments found that overexpression of mmaA1 in M. tuberculosis resulted in accumulation of trans-unsaturated and -cyclopropanated oxygenated mycolates (27). These data suggested that MmaA1 acts in the biosynthesis of trans-cyclopropanated oxygenated mycolates either by adding the methyl branch distal to the cyclopropane ring or as a cis-trans isomerase or both. In addition, although there was a defect in cis cyclopropanation of methoxymycolates in the ΔmmaA2 strain, this defect was mild, suggesting redundancy with another unidentified enzyme. In this article, we define novel functions for three cyclopropane synthases using a new selectable marker to construct M. tuberculosis strains deficient in multiple mycolic acid methyltransferases. Through this approach, we show that CmaA2 and MmaA2 are redundant for cis cyclopropanation of the proximal position of the methoxymycolates and ketomycolates and that MmaA1 is upstream of CmaA2 in trans cyclopropanation.     

Allosteric Transitions of Supramolecular Systems Explored by Network Models: Application to Chaperonin GroEL

Identification of pathways involved in the structural transitions of biomolecular systems is often complicated by the transient nature of the conformations visited across energy barriers and the multiplicity of paths accessible in the multidimensional energy landscape. This task becomes even more challenging in exploring molecular systems on the order of megadaltons. Coarse-grained models that lend themselves to analytical solutions appear to be the only possible means of approaching such cases. Motivated by the utility of elastic network models for describing the collective dynamics of biomolecular systems and by the growing theoretical and experimental evidence in support of the intrinsic accessibility of functional substates, we introduce a new method, adaptive anisotropic network model (aANM), for exploring functional transitions. Application to bacterial chaperonin GroEL and comparisons with experimental data, results from action minimization algorithm, and previous simulations support the utility of aANM as a computationally efficient, yet physically plausible, tool for unraveling potential transition pathways sampled by large complexes/assemblies. An important outcome is the assessment of the critical inter-residue interactions formed/broken near the transition state(s), most of which involve conserved residues.     

Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis

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