Structural Analyses of the Multicopper Site of CopG Support a Role as a Redox Enzyme.

Metal ions can be both essential components of cells as well as potential toxins if present in excess. Organisms utilize a variety of protein systems to maintain the concentration of metal ions within the appropriate range for cellular function, and to avoid concentrations where cellular damage can occur. In bacteria, numerous proteins contribute to copper homeostasis, including copper transporters, chelators, and redox enzymes. The genes that encode these proteins are often found in clusters, thus providing modular components that work together to achieve homeostasis. A better understanding of how these components function and cooperate to achieve metal ion resistance is needed, given the extensive use of metal ions, including copper, as broad-spectrum biocides in a variety of clinical and environmental settings. The copG gene is a common component of such copper resistance clusters, but its contribution to copper resistance is not well understood. In this review the available information about the CopG protein encoded by this gene is summarized. Comparison of the recent structure to diverse copper-containing metallochaperones, metalloenzymes, and electron transfer proteins suggests that CopG is a redox enzyme that uses multiple copper ions as active site redox cofactors to act on additional copper ion substrates. Mechanisms for both oxidase and reductase activity are proposed, and the biological advantages that these activities can contribute in conjunction with existing systems are described.

The bacterial copper resistance protein CopG contains a cysteine-bridged tetranuclear copper cluster.

CopG is an uncharacterized protein ubiquitous in Gram-negative bacteria whose gene frequently occurs in clusters of copper resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system.

Conditionally disordered proteins: bringing the environment back into the fold.

For many proteins, biological function requires the folding of the polypeptide chain into a unique and persistent tertiary structure. This review concerns proteins that adopt a specific tertiary structure to function, but are otherwise partially or completely disordered. The biological cue for protein folding is environmental perturbation or minor post-translational modification. Hence, we term these proteins conditionally disordered. Many of these proteins recognize and bind other molecules, and conditional disorder has been hypothesized to allow for more nuanced control and regulation of binding processes. However, this remains largely unproven. The sequences of conditionally disordered proteins suggest their propensity to fold; yet, under the standard laboratory conditions, they do not do so, which may appear surprising. We argue that the surprise results from the failure to consider the role of the environment in protein structure formation and that conditional disorder arises as a natural consequence of the marginal stability of the folded state.

Model for coupled insertion and folding of membrane-spanning proteins.

Current understanding of the forces directing the folding of integral membrane proteins is very limited compared to the detailed picture available for water-soluble proteins. While mechanistic studies of the folding process in vitro have been conducted for only a small number of membrane proteins, the available evidence indicates that their folding process is thermodynamically driven like that of soluble proteins. In vivo, however, the majority of integral membrane proteins are installed in membranes by dedicated machinery, suggesting that the cellular systems may act to facilitate and regulate the spontaneous physical process of folding. Both the in vitro folding process and the in vivo pathway must navigate an energy landscape dominated by the energetically favorable burial of hydrophobic segments in the membrane interior and the opposition to folding due to the need for passage of polar segments across the membrane. This manuscript describes a simple, exactly solvable model which incorporates these essential features of membrane protein folding. The model is used to compare the folding time under conditions which depict both the in vitro and in vivo pathways. It is proposed that the cellular complexes responsible for insertion of membrane proteins act by lowering the energy barrier for passage of polar regions through the membrane, thereby allowing the chain to more rapidly achieve the folded state.

YC-1 binding to the ß subunit of soluble guanylyl cyclase overcomes allosteric inhibition by the α subunit.

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remain unknown. Using linked equilibrium binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the ß subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 µM, depending on the construct. In the presence of CO, these values decrease to 0.6-1.1 µM. Pfizer compound 25 bound ∼10-fold weaker than YC-1 in the absence of CO, whereas compound BAY 41-2272 bound particularly tightly in the presence of CO (Kd = 30-90 nM). Additionally, we found that CO binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 µM) than to the isolated heme domain (Kd = 0.2 µM for Manduca ß H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ∼ 1 µM). These data indicate the α subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the α subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

Zinc enhances adiponectin oligomerization to octadecamers but decreases the rate of disulfide bond formation.

Adiponectin, a hormone secreted from adipocytes, has been shown to protect against development of insulin resistance, ischemia-reperfusion injury, and inflammation. Adiponectin assembles into multiple oligomeric isoforms: trimers, hexamers and several higher molecular weight (HMW) species. Of these, the HMW species are selectively decreased during the onset of type 2 diabetes. Despite the critical role of HMW adiponectin in insulin responsiveness, its assembly process is poorly understood. In this report, we investigated the role of divalent cations in adiponectin assembly. Purified adiponectin 18mers, the largest HMW species, did not collapse to smaller oligomers after treatment with high concentrations of EDTA. However, treatment with EDTA or another chelator DTPA inhibited the oligomerization of 18mers from trimers in vitro. Zn(2+) specifically increased the formation of 18mers when compared with Cu(2+), Mg(2+), and Ca(2+). Distribution of adiponectin oligomers secreted from zinc chelator TPEN-treated rat adipocytes skewed toward increased proportions of hexamers and trimers. While we observed presence of zinc in adiponectin purified from calf serum, the role of zinc in disulfide bonding between oligomers was examined because the process is critical for 18mer assembly. Surprisingly, Zn(2+) inhibited disulfide bond formation early in the oligomerization process. We hypothesize that initial decreases in disulfide formation rates could allow adiponectin subunits to associate before becoming locked in fully oxidized conformations incapable of further oligomerization. These data demonstrate that zinc stimulates oligomerization of HMW adiponectin and possibly other disulfide-dependent protein assembly processes.

Disulfide-dependent self-assembly of adiponectin octadecamers from trimers and presence of stable octadecameric adiponectin lacking disulfide bonds in vitro.

Adiponectin is a circulating insulin-sensitizing hormone that homooligomerizes into trimers, hexamers, and higher molecular weight (HMW) species. Low levels of circulating HMW adiponectin appear to increase the risk for insulin resistance. Currently, assembly of adiponectin oligomers and, consequently, mechanisms responsible for decreased HMW adiponectin in insulin resistance are not well understood. In the work reported here, we analyzed the reassembly of the most abundant HMW adiponectin species, the octadecamer, following its collapse to smaller oligomers in vitro. Purified bovine serum adiponectin octadecamer was treated with reducing agents at pH 5 to obtain trimers. These reduced trimers partially and spontaneously reassembled into octadecamers upon oxidative formation of disulfide bonds. Disulfide bonds appear to occupy a greater role in the process of oligomerization than in the structural stabilization of mature octadecamer. Stable octadecamers lacking virtually all disulfide bonds could be observed in abundance using native gel electrophoresis, dynamic light scattering, and collision-induced dissociation nanoelectrospray ionization mass spectrometry. These findings indicate that while disulfide bonds help to maintain the mature octadecameric adiponectin structure, their more important function is to stabilize intermediates during the assembly of octadecamer. Adiponectin oligomerization must proceed through intermediates that are at least partially reduced. Accordingly, fully oxidized adiponectin hexamers failed to reassemble into octadecamers at a rate comparable to that of reduced trimers. As the findings from the present study are based on in vitro experiments, their in vivo relevance remains unclear. Nevertheless, they describe a redox environment-dependent model of adiponectin oligomerization that can be tested using cell-based approaches.

A robust approach for analyzing a heterogeneous structural ensemble.

Intrinsically unstructured proteins (IUP) are widespread in eukaryotes and participate in numerous cellular processes, but a structural explanation of the mechanisms they use to recognize and bind their diverse targets has proved elusive. Transcriptional activator domains are one class of IUP that function by recruiting other factors into basal transcription complexes. Transcriptional activator domains are known to use electrostatic interactions for recognition, but it is unclear how this could be accomplished by a structurally heterogeneous ensemble. To investigate this question, we performed principal component analysis on the atomic contact maps of an experimentally restrained ensemble of the human p53 transcriptional activator domain. This analysis revealed that the ensemble is conspicuously nonrandom and permitted a straightforward identification of persistent structural features and their relative probabilities. It was observed that six predominant long-range contacts are combinatorially arranged in 13 clusters of structures. Potential surfaces of the aligned clusters showed that these contacts uniformly organize the negative charges of the highly acidic p53 transcriptional activator domain on one face of the clusters. This observation provides a structural basis for the recruitment of other factors into basal transcription complexes and further supports the hypothesis that the structural ensembles of IUPs are not random and instead have evolved under selection to maintain specific structural features.

Chirality of coiled coils: elasticity matters.

Coiled coils are important protein-protein interaction motifs with high specificity that are used to assemble macromolecular complexes. Their simple geometric organization, consisting of alpha helices wrapped around each other, confers remarkable mechanical properties. A geometrical and mechanical continuous model taking into account sequence effects and based on the superhelical winding of the constituent helices is introduced, and a continuous family of solutions in which the oligomerization interactions are satisfied is derived. From these solutions, geometric and structural properties, such as the chirality and pitch of the coiled coil and the location of residues, are obtained. The theoretical predictions are compared to x-ray data from the leucine zipper motif.

Periplasmic metal-resistance protein CusF exhibits high affinity and specificity for both CuI and AgI.

The periplasmic protein CusF, as a part of the CusCFBA efflux complex, plays a role in resistance to elevated levels of copper and silver in Escherichia coli. Although homologues have been identified in other Gram-negative bacteria, the substrate of CusF and its precise role in metal resistance have not been described. Here, isothermal titration calorimetry (ITC) was used to demonstrate that CusF binds with high affinity to both Cu(I) and Ag(I) but not Cu(II). The affinity of CusF for Ag(I) was higher than that for Cu(I), which could reflect more efficient detoxification of Ag(I) given the lack of a cellular need for Ag(I). The chemical shifts in the nuclear magnetic resonance (NMR) spectra of CusF-Ag(I) as compared to apo-CusF show that the region of CusF most affected by Ag(I) binding encompasses three absolutely conserved residues: H36, M47, and M49. This suggests that these residues may play a role in Ag(I) coordination. The NMR spectra of CusF in the presence of Cu(II) do not indicate specific binding, which is in agreement with the ITC data. We conclude that Cu(I) and Ag(I) are the likely physiological substrates.

A kinetic theory of tertiary contact formation coupled to the helix-coil transition in polypeptides.

The framework model and the hydrophobic collapse model represent two canonical descriptions of the protein folding process. The first places primary reliance on the short-range interactions of secondary structure and the second assigns greater importance to the long-range interactions of tertiary structure. The availability of increasingly detailed information about the folding mechanisms of diverse proteins suggests that both are important and the folding mechanism of most proteins utilizes different combinations of such interactions. A prior report described the XHC model, an extended helix-coil theory, which treats the mutual stabilization of secondary and tertiary structure in simple alpha-helical proteins at equilibrium. In this study, a kinetic scheme describing tertiary contact formation has been developed which relaxes to the XHC equilibrium model. The relaxation is governed by the relative stabilities of the equilibrium states and an additional factor which represents an activation energy for formation of a tertiary contact. The model can be used to simulate time-dependent properties of the ensemble of conformations during the entire folding process, and the resulting predictions are applicable to a range of experimental methods. This XHC kinetic model enables investigation of the relative influence of secondary and tertiary interactions on folding mechanisms.

High polar content of long buried blocks of sequence in protein domains suggests selection against amyloidogenic non-polar sequences.

Native protein structures achieve stability in part by burying hydrophobic side-chains. About 75% of all amino acid residues buried in protein interiors are non-polar. Buried residues are not uniformly distributed in protein sequences, but sometimes cluster as contiguous polypeptide stretches that run through the interior of protein domain structures. Such regions have an intrinsically high local sequence density of non-polar residues, creating a potential problem: local non-polar sequences also promote protein misfolding and aggregation into non-native structures such as the amyloid fibrils in Alzheimer's disease. Here we show that long buried blocks of sequence in protein domains of known structure have, on average, a lower content of non-polar amino acids (about 70%) than do isolated buried residues (about 80%). This trend is observed both in small and in large protein domains and is independent of secondary structure. Long, completely non-polar buried stretches containing many large side-chains are particularly avoided. Aspartate residues that are incorporated in long buried stretches were found to make fewer polar interactions than those in short stretches, hinting that they may be destabilizing to the native state. We suggest that evolutionary pressure is acting on non-native properties, causing buried polar residues to be placed at positions where they would break up aggregation-prone non-polar sequences, perhaps even at some cost to native state stability.

A model for the coupling of alpha-helix and tertiary contact formation.

Peptides corresponding to excised alpha-helical segments of natural proteins can spontaneously form helices in solution. However, peptide helices are usually substantially less stable in solution than in the structural context of a folded protein, because of the additional interactions possible between helices in a protein. Such interactions can be thought of as coupling helix formation and tertiary contact formation. The relative energetic contributions of the two processes to the total energy of the folded state of a protein is a matter of current debate. To investigate this balance, an extended helix-coil model (XHC) that incorporates both effects has been constructed. The model treats helix formation with the Lifson-Roig formalism, which describes helix initiation and propagation through cooperative local interactions. The model postulates an additional parameter representing participation of a site in a tertiary contact. In the model, greater helix stability can be achieved through combinations of these short-range and long-range interactions. For instance, stronger tertiary contacts can compensate for helices with little intrinsic stability. By varying the strength of the nonlocal interactions, the model can exhibit behavior consistent with a variety of qualitative models describing the relative importance of secondary and tertiary structure. Moreover, the model is explicit in that it can be used to fit experimental data to individual peptide sequences, providing a means to quantify the two contributions on a common energetic basis.

Fundamental processes of protein folding: measuring the energetic balance between helix formation and hydrophobic interactions.

Theories of protein folding often consider contributions from three fundamental elements: loops, hydrophobic interactions, and secondary structures. The pathway of protein folding, the rate of folding, and the final folded structure should be predictable if the energetic contributions to folding of these fundamental factors were properly understood. alphatalpha is a helix-turn-helix peptide that was developed by de novo design to provide a model system for the study of these important elements of protein folding. Hydrogen exchange experiments were performed on selectively 15N-labeled alphatalpha and used to calculate the stability of hydrogen bonds within the peptide. The resulting pattern of hydrogen bond stability was analyzed using a version of Lifson-Roig model that was extended to include a statistical parameter for tertiary interactions. This parameter, x, represents the additional statistical weight conferred upon a helical state by a tertiary contact. The hydrogen exchange data is most closely fit by the XHC model with an x parameter of 9.25. Thus the statistical weight of a hydrophobic tertiary contact is approximately 5.8x the statistical weight for helix formation by alanine. The value for the x parameter derived from this study should provide a basis for the understanding of the relationship between hydrophobic cluster formation and secondary structure formation during the early stages of protein folding.

Repeat protein architectures predicted by a continuum representation of fold space.

It is an open question whether nature has utilized all possible protein folds. For a simple protein architecture, the helical repeats, we report a method to address this question based on a mapping between the set of repetitive curves and a space of parameters specifying the curve. The exploration of the parameter space for a particular architecture enables a systematic exploration of the fold space for that protein architecture. In a planar subspace of the parameter space of helical repeats we have identified points corresponding to both naturally occurring folds and potential folds not observed so far.

Structure of the constitutively active double mutant CheYD13K Y106W alone and in complex with a FliM peptide.

CheY is a member of the response regulator protein superfamily that controls the chemotactic swimming response of motile bacteria. The CheY double mutant D13K Y106W (CheY**) is resistant to phosphorylation, yet is a highly effective mimic of phosphorylated CheY in vivo and in vitro. The conformational attributes of this protein that enable it to signal in a phosphorylation-independent manner are unknown. We have solved the crystal structure of selenomethionine-substituted CheY** in the presence of its target, a peptide (FliM16) derived from the flagellar motor switch, FliM, to 1.5A resolution with an R-factor of 19.6%. The asymmetric unit contains four CheY** molecules, two with FliM16 bound, and two without. The two CheY** molecules in the asymmetric unit that are bound to FliM16 adopt a conformation similar to BeF3- -activated wild-type CheY, and also bind FliM16 in a nearly identical manner. The CheY** molecules that do not bind FliM16 are found in a conformation similar to unphosphorylated wild-type CheY, suggesting that the active phenotype of this mutant is enabled by a facile interconversion between the active and inactive conformations. Finally, we propose a ligand-binding model for CheY and CheY**, in which Ile95 changes conformation in a Tyr/Trp106-dependent manner to accommodate FliM.

The identity of proteins associated with a small heat shock protein during heat stress in vivo indicates that these chaperones protect a wide range of cellular functions.

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and co-chaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function.

Thermolysin in the absence of substrate has an open conformation.

The bacterial neutral proteases have been proposed to undergo hinge-bending during their catalytic cycle. However, in thermolysin, the prototypical member of the family, no significant conformational change has been observed. The structure of thermolysin has now been determined in a new crystal form that for the first time shows the enzyme in the absence of a ligand bound in the active site. This is shown to be an 'open' form of the enzyme. The relative orientation of the two domains that define the active-site cleft differ by a 5 degrees rotation relative to their positions in the previously studied ligand-bound 'closed' form. Based on structural comparisons, kinetic studies on mutants and molecular-dynamics simulations, Gly78 and Gly135-Gly136 have previously been suggested as two possible hinge regions. Comparison of the 'open' and 'closed' structures suggests that neither of the proposed hinge regions completely accounts for the observed displacement. The concerted movement of a group of side chains suggested to be associated with the hinge-bending motion is, however, confirmed.