STRUCTURE AND FUNCTION OF THE GROUP III CHAPERONINS, A UNIQUE CLADE OF PROTEIN FOLDING NANOMACHINES

The survival and descent of cells is universally dependent on maintaining their proteins in a properly folded condition. It is widely accepted that the information for the folding of the nascent polypeptide chain into a native protein is encrypted in the amino acid sequence, and the Nobel Laureate Christian Anfinsen was the first to demonstrate that a protein could spontaneously refold after complete unfolding. However, it became clear that the observed folding rates for many proteins were much slower than rates estimated in vivo. This led to the recognition of required protein-protein interactions that promote proper folding. A unique group of proteins, the molecular chaperones, are responsible for maintaining protein homeostasis during normal growth as well as stress conditions. Chaperonins (CPNs) are ubiquitous and essential chaperones. They form ATP-dependent, hollow complexes that encapsulate polypeptides in two back-to-back stacked multisubunit rings, facilitating protein folding through highly cooperative allosteric articulation. CPNs are usually classified into Group I and Group II. Here, I report the characterization of a novel CPN belonging to a third Group, recently discovered in bacteria. Group III CPNs have close phylogenetic association to the Group II CPNs found in Archaea and Eukarya, and may be a relic of the Last Common Ancestor of the CPN family. The gene encoding the Group III CPN from Carboxydothermus hydrogenoformans and Candidatus Desulforudis audaxviator was cloned in E. coli and overexpressed in order to both characterize the protein and to demonstrate its ability to function as an ATPase chaperone. The opening and closing cycle of the Chy chaperonin was examined via site-directed mutations affecting the ATP binding site at R155. To relate the mutational analysis to the structure of the CPN, the crystal structure of both the AMP-PNP (an ATP analogue) and ADP bound forms were obtained in collaboration with Sun-Shin Cha in Seoul, South Korea. The ADP and ATP binding site substitutions resulted in frozen forms of the structures in open and closed conformations. From this, mutants were designed to validate hypotheses regarding key ATP interacting sites as well as important stabilizing interactions, and to observe the physical properties of the resulting complexes by calorimetry.

Molecular interactions: metal ions and protein chaperones in the urease system from Helicobacter pylori

Although nickel is a toxic metal for living organisms in its soluble form, its importance in many biological processes recently emerged. In this view, the investigation of the nickel-dependent enzymes urease and [NiFe]-hydrogenase, especially the mechanism of nickel insertion into their active sites, represent two intriguing case studies to understand other analogous systems and therefore to lead to a comprehension of the nickel trafficking inside the cell. Moreover, these two enzymes have been demonstrated to ensure survival and colonization of the human pathogen H. pylori, the only known microorganism able to proliferate in the gastric niche. The right nickel delivering into the urease active site requires the presence of at least four accessory proteins, UreD, UreE, UreF and UreG. Similarly, analogous process is principally mediated by HypA and HypB proteins in the [NiFe]-hydrogenase system. Indeed, HpHypA and HpHypB also have been proposed to act in the activation of the urease enzyme from H. pylori, probably mobilizing nickel ions from HpHypA to the HpUreE-HpUreG complex. A complete comprehension of the interaction mechanism between the accessory proteins and the crosstalk between urease and hydrogenase accessory systems requires the determination of the role of each protein chaperone that strictly depends on their structural and biochemical properties. The availability of HpUreE, HpUreG and HpHypA proteins in a pure form is a pre-requisite to perform all the subsequent protein characterizations, thus their purification was the first aim of this work. Subsequently, the structural and biochemical properties of HpUreE were investigated using multi-angle and quasi-elastic light scattering, as well as NMR and circular dichroism spectroscopy. The thermodynamic parameters of Ni2+ and Zn2+ binding to HpUreE were principally established using isothermal titration calorimetry and the importance of key histidine residues in the process of binding metal ions was studied using site-directed mutagenesis. The molecular details of the HpUreE-HpUreG and HpUreE-HpHypA protein-protein assemblies were also elucidated. The interaction between HpUreE and HpUreG was investigated using ITC and NMR spectroscopy, and the influence of Ni2+ and Zn2+ metal ions on the stabilization of this association was established using native gel electrophoresis, light scattering and thermal denaturation scanning followed by CD spectroscopy. Preliminary HpUreE-HpHypA interaction studies were conducted using ITC. Finally, the possible structural architectures of the two protein-protein assemblies were rationalized using homology modeling and docking computational approaches. All the obtained data were interpreted in order to achieve a more exhaustive picture of the urease activation process, and the correlation with the accessory system of the hydrogenase enzyme, considering the specific role and activity of the involved protein players. A possible function for Zn2+ in the chaperone network involved in Ni2+ trafficking and urease activation is also envisaged.

Immunophilin chaperones in steroid receptor signalling

The immunophilin cochaperones, cyclophilin 40 (CyP40), FKBP51 and FKBP52 and PP5, a serine/threonine protein phosphatase, have been implicated as modulators of steroid receptor function through their association with Hsp90, a molecular chaperone with a key role in steroid hormone signalling. Although progress towards a satisfying definition for the role of these components in steroid receptor complexes has been slow, recent developments arising from novel approaches in both yeast and mammalian systems, together with available crystal structures for Hsp90 and some of these cochaperones, are beginning to provide important clues about their function. Hsp90, recently identified as a member of the GHKL superfamily of ATPases, is the central player in receptor assembly, an energy-driven process that allows receptor and the immunophilins to be proximally located, or to interact directly, on a Hsp90 scaffold. Immunophilin structure, relative abundance, their binding affinity for Hsp90 and their ability to interact with specific receptors may all contribute to a selective preference of the immunophilins for individual receptors. Association of receptors with different immunophilins leads to differential functional consequences for receptor activity. Observations of glucocorticoid resistance in New World primates, attributed to FKBP51 overexpression and incorporation into glucocorticoid receptor complexes, have provided the first evidence that these cochaperones can control hormone-binding affinity. Application of a yeast model to FKBP52 function in the glucocorticoid receptor system has now provided crucial evidence that this immunophilin enhances receptor transcriptional activity by increasing receptor avidity for hormone through PPIase-mediated conformational changes in the ligand-binding domain. A recent novel finding suggests that hormone binding may induce a functional exchange of immunophilins in receptor complexes and that the modified complex directs receptor to the nucleus.

Active solubilization and refolding of stable protein aggregates by cooperative unfolding action of individual hsp70 chaperones.

Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

A system-level, molecular evolutionary analysis of mammalian phototransduction

Visual perception is initiated in the photoreceptor cells of the retina via the phototransduction system.This system has shown marked evolution during mammalian divergence in such complex attributes as activation time and recovery time. We have performed a molecular evolutionary analysis of proteins involved in mammalianphototransduction in order to unravel how the action of natural selection has been distributed throughout thesystem to evolve such traits. We found selective pressures to be non-randomly distributed according to both a simple protein classification scheme and a protein-interaction network representation of the signaling pathway. Proteins which are topologically central in the signaling pathway, such as the G proteins, as well as retinoid cycle chaperones and proteins involved in photoreceptor cell-type determination, were found to be more constrained in their evolution. Proteins peripheral to the pathway, such as ion channels and exchangers, as well as the retinoid cycle enzymes, have experienced a relaxation of selective pressures. Furthermore, signals of positive selection were detected in two genes: the short-wave (blue) opsin (OPN1SW) in hominids and the rod-specific Na+/Ca2+,K+ ion exchanger (SLC24A1) in rodents. The functions of the proteins involved in phototransduction and the topology of the interactions between them have imposed non-random constraints on their evolution. Thus, in shaping or conserving system-level phototransduction traits, natural selection has targeted the underlying proteins in a concerted manner.

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

Crystal Structures of Xanthomonas Small Heat Shock Protein Provide a Structural Basis for an Active Molecular Chaperone Oligomer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FHR127H) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-gamma were able to synthesize FHR127H, the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-gamma secrete FH without retention in the ER. Moreover, the retention of FHR127H provoked enlargement of ER cisterns after treatment with IFN-gamma. A similar ER retention was observed in Cos-7 cells expressing the mutant FHR127H protein. Despite this deficiency in secretion, we show that the FHR127H mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein. The Journal of Immunology, 2012, 189: 3242-3248.

Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.

Molecular and cell biology aspects of plague

A 70-kb virulence plasmid (sometimes called pYV) enables Yersinia spp. to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, a system consisting of secreted proteins called Yops and their dedicated type III secretion apparatus called Ysc. The Ysc apparatus forms a channel composed of 29 proteins. Of these, 10 have counterparts in almost every type III system. Secretion of some Yops requires the assistance, in the bacterial cytosol, of small individual chaperones called the Syc proteins. These chaperones act as bodyguards or secretion pilots for their partner Yop. Yop proteins fall into two categories. Some are intracellular effectors, whereas the others are “translocators” needed to deliver the effectors across the eukaryotic plasma membrane, into eukaryotic cells. The translocators (YopB, YopD, LcrV) form a pore of 16–23 Å in the eukaryotic cell plasma membrane. The effector Yops are YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT. YopH is a powerful phosphotyrosine phosphatase playing an antiphagocytic role by dephosphorylating several focal adhesion proteins. YopE and YopT contribute to antiphagocytic effects by inactivating GTPases controlling cytoskeleton dynamics. YopP/YopJ plays an anti-inflammatory role by preventing the activation of the transcription factor NF-κB. It also induces rapid apoptosis of macrophages. Less is known about the role of the phosphoserine kinase YopO/YpkA and YopM.

Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

Analysis of Molecular Dynamics Simulations of Protein Folding

Microsecond long Molecular Dynamics (MD) trajectories of biomolecular processes are now possible due to advances in computer technology. Soon, trajectories long enough to probe dynamics over many milliseconds will become available. Since these timescales match the physiological timescales over which many small proteins fold, all atom MD simulations of protein folding are now becoming popular. To distill features of such large folding trajectories, we must develop methods that can both compress trajectory data to enable visualization, and that can yield themselves to further analysis, such as the finding of collective coordinates and reduction of the dynamics. Conventionally, clustering has been the most popular MD trajectory analysis technique, followed by principal component analysis (PCA). Simple clustering used in MD trajectory analysis suffers from various serious drawbacks, namely, (i) it is not data driven, (ii) it is unstable to noise and change in cutoff parameters, and (iii) since it does not take into account interrelationships amongst data points, the separation of data into clusters can often be artificial. Usually, partitions generated by clustering techniques are validated visually, but such validation is not possible for MD trajectories of protein folding, as the underlying structural transitions are not well understood. Rigorous cluster validation techniques may be adapted, but it is more crucial to reduce the dimensions in which MD trajectories reside, while still preserving their salient features. PCA has often been used for dimension reduction and while it is computationally inexpensive, being a linear method, it does not achieve good data compression. In this thesis, I propose a different method, a nonmetric multidimensional scaling (nMDS) technique, which achieves superior data compression by virtue of being nonlinear, and also provides a clear insight into the structural processes underlying MD trajectories. I illustrate the capabilities of nMDS by analyzing three complete villin headpiece folding and six norleucine mutant (NLE) folding trajectories simulated by Freddolino and Schulten [1]. Using these trajectories, I make comparisons between nMDS, PCA and clustering to demonstrate the superiority of nMDS. The three villin headpiece trajectories showed great structural heterogeneity. Apart from a few trivial features like early formation of secondary structure, no commonalities between trajectories were found. There were no units of residues or atoms found moving in concert across the trajectories. A flipping transition, corresponding to the flipping of helix 1 relative to the plane formed by helices 2 and 3 was observed towards the end of the folding process in all trajectories, when nearly all native contacts had been formed. However, the transition occurred through a different series of steps in all trajectories, indicating that it may not be a common transition in villin folding. The trajectories showed competition between local structure formation/hydrophobic collapse and global structure formation in all trajectories. Our analysis on the NLE trajectories confirms the notion that a tight hydrophobic core inhibits correct 3-D rearrangement. Only one of the six NLE trajectories folded, and it showed no flipping transition. All the other trajectories get trapped in hydrophobically collapsed states. The NLE residues were found to be buried deeply into the core, compared to the corresponding lysines in the villin headpiece, thereby making the core tighter and harder to undo for 3-D rearrangement. Our results suggest that the NLE may not be a fast folder as experiments suggest. The tightness of the hydrophobic core may be a very important factor in the folding of larger proteins. It is likely that chaperones like GroEL act to undo the tight hydrophobic core of proteins, after most secondary structure elements have been formed, so that global rearrangement is easier. I conclude by presenting facts about chaperone-protein complexes and propose further directions for the study of protein folding.

Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.