The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase.

The kinetic mechanism of the oxidative decarboxylation of 2R,3S-isopropylmalate by the NAD-dependent isopropylmalate dehydrogenase of Thermus thermophilus was investigated. Initial rate results typical of random or steady-state ordered sequential mechanisms are obtained for both the wild-type and two mutant enzymes (E87G and E87Q) regardless of whether natural or alternative substrates (2R-malate, 2R,3S-tartrate and/or NADP) are utilized. Initial rate data fail to converge on a rapid equilibrium-ordered pattern despite marked reductions in specificity (kcat/Km) caused by the mutations and alternative substrates. Although the inhibition studies alone might suggest an ordered kinetic mechanism with cofactor binding first, a detailed analysis reveals that the expected noncompetitive patterns appear uncompetitive because the dissociation constants from the ternary complexes are far smaller than those from the binary complexes. Equilibrium fluorescence studies both confirm the random binding of substrates and the kinetic estimates of the dissociation constants of the substrates from the binary complexes. The latter are not distributed markedly by the mutations at site 87. Mutations at site 87 do not affect the dissociation constants from the binary complexes, but do greatly increase the Michaelis constants, indicating that E87 helps stabilize the Michaelis complex of the wild-type enzyme. The available structural data, the patterns of the kinetics results, and the structure of a pseudo-Michaelis complex of the homologous isocitrate dehydrogenase of Escherichia coli suggest that E87 interacts with the nicotinamide ring.     

Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate

Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central alpha-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with DeltaG degrees (H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.     

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

Intermediates and the folding of proteins L and G

We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted beta-1 and beta-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding, and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third beta-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally, the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first-order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment.     

Solid-state NMR as a method to reveal structure and membrane-interaction of amyloidogenic proteins and peptides

It is important to understand the Amyloid fibril formation in view of numerous medical and biochemical aspects. Structural determination of amyloid fibril has been extensively studied using electron microscopy. Subsequently, solid state NMR spectroscopy has been realized to be the most important means to determine not only microscopic molecular structure but also macroscopic molecular packing. Molecular structure of amyloid fibril was first predicted to be parallel β-sheet structure, and subsequently, was further refined for Aβ(1-40) to be cross β-sheet with double layered in register parallel β-sheet structure by using solid state NMR spectroscopy. On the other hand, anti-parallel β-sheet structure has been reported to short fragments of Aβ-amyloid and other amyloid forming peptides. Kinetic study of amyloid fibril formation has been studied using a variety of methods, and two-step autocatalytic reaction mechanism used to explain fibril formation. Recently, stable intermediates or proto-fibrils have been observed by electron microscope (EM) images. Some of the intermediates have the same microscopic structure as the matured fibril and subsequently change to matured fibrils. Another important study on amyloid fibril formation is determination of the interaction with lipid membranes, since amyloid peptide are cleaved from amyloid precursor proteins in the membrane interface, and it is reported that amyloid lipid interaction is related to the cytotoxicity. Finally it is discussed how amyloid fibril formation can be inhibited. Firstly, properly designed compounds are reported to have inhibition ability of amyloid fibril formation by interacting with amyloid peptide. Secondly, it is revealed that site directed mutation can inhibit amyloid fibril formation. These inhibitors were developed by knowing the fibril structure determined by solid state NMR.     

Population analyses of kinetic partitioning in protein folding

A simple lattice model of protein folding is studied in order to analyze the kinetic partitioning phenomena in the energy landscape perspective. By restricting the area of conformational space, it becomes possible to follow many Monte Carlo trajectories until they reach equilibrium. Alteration of population of trajectories is monitored and the relations between the energy landscape and kinetics are examined. Kinetic partitioning phenomena are categorized into different types in terms of characteristic time constants and partitioning ratio. In a specific partitioning process, refolding proceeds along the parallel pathways; the time constants have a temperature dependence similar to that observed in hen lysozyme. High-energy conformations are classified into groups according to the probability that the trajectories starting from those conformations will reach each energy valley. The partitioning ratio is determined by the way in which the conformational space is organized into these groups.     

Novel tertiary structures and stereospecificities of periplasmic binding proteins involved in active transport and chemotaxis

Binding proteins, which are located in the periplasmic space of Gram-negative bacteria, are essential components of osmotic shock-sensitive active transport systems and Chemotaxis. Described briefly herein are the high resolution molecular structures of four binding proteins specific for (i) L-arabinose, (ii) sulphate, (iii) D-galactose, and (iv) leucine, isoleucine or valine which we have recently determined. The first three proteins contained bound substrates. Several novel substrate binding properties of the arabinose- and sulphate-binding proteins as revealed by structure refinement at 1.7 Å resolution are also presented. These results have profound significance in understanding both protein structures and substrate-protein interactions.     

Structures of Ric-8B in complex with Gα protein folding clients reveal isoform specificity mechanisms

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Smoothing molecular interactions: the "kinetic buffer" effect of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) widely participate in molecular recognition and signaling processes in cells by interacting with other molecules. Compared with ordered proteins, IDPs usually possess stronger intermolecular interactions in binding. As a result, the interface structure of IDPs in complexes is distinct from that of ordered-protein complexes, and this difference may have essential effect on the response to various perturbations in a cell. In this study, we examined the perturbations of intermolecular interactions and temperature on the coupled folding and binding processes of pKID to KIX domains by performing molecular dynamics simulations. By comparing a series of virtual pKID systems with various degree of disorder, we found that the complex stability and the binding kinetics of the disordered systems were less sensitive to the perturbations than the ordered systems. The origin of the lower response sensitivity of IDPs was attributed to their higher flexibility in the complex interface, which was further supported by an analysis on protein complex structures. On the basis of our simulations and results from the literature, we speculate IDPs may not only interact with their biological partners with high specificity and low affinity but also may be resistant to the perturbations in the environment and transmit signals fast and smooth. We proposed to name it the "kinetic buffer" effect.     

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.     

Computational design, construction, and characterization of a set of specificity determining residues in protein-protein interactions

Proteins interact with different partners to perform different functions and it is important to elucidate the determinants of partner specificity in protein complex formation. Although methods for detecting specificity determining positions have been developed previously, direct experimental evidence for these amino acid residues is scarce, and the lack of information has prevented further computational studies. In this article, we constructed a dataset that is likely to exhibit specificity in protein complex formation, based on available crystal structures and several intuitive ideas about interaction profiles and functional subclasses. We then defined a “structure‐based specificity determining position (sbSDP)” as a set of equivalent residues in a protein family showing a large variation in their interaction energy with different partners. We investigated sequence and structural features of sbSDPs and demonstrated that their amino acid propensities significantly differed from those of other interacting residues and that the importance of many of these residues for determining specificity had been verified experimentally. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction

Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.     

Differential use of two AP-3-mediated pathways by lysosomal membrane proteins

The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.     

Structures of p38alpha active mutants reveal conformational changes in L16 loop that induce autophosphorylation and activation

p38 mitogen-activated protein (MAP) kinases function in numerous signaling processes and are crucial for normal functions of cells and organisms. Abnormal p38 activity is associated with inflammatory diseases and cancers making the understanding of its activation mechanisms highly important. p38s are commonly activated by phosphorylation, catalyzed by MAP kinase kinases (MKKs). Moreover, it was recently revealed that the p38alpha is also activated via alternative pathways, which are MKK independent. The structural basis of p38 activation, especially in the alternative pathways, is mostly unknown. This lack of structural data hinders the study of p38's biology as well as the development of novel strategies for p38 inhibition. We have recently discovered and optimized a novel set of intrinsically active p38 mutants whose activities are independent of any upstream activation. The high-resolution crystal structures of the intrinsically active p38alpha mutants reveal that local alterations in the L16 loop region promote kinase activation. The L16 loop can be thus regarded as a molecular switch that upon conformational changes promotes activation. We suggest that similar conformational changes in L16 loop also occur in natural activation mechanisms of p38alpha in T-cells. Our biochemical studies reveal novel mechanistic insights into the activation process of p38. In this regard, the results indicate that the activation mechanism of the mutants involves dimerization and subsequent trans autophosphorylation on Thr180 (on the phosphorylation lip). Finally, we suggest a model of in vivo p38alpha activation induced by the L16 switch with auto regulatory characteristics.     

Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase

During the final stages of cell-wall synthesis in bacteria, penicillin-binding proteins (PBPs) catalyse the cross-linking of peptide chains from adjacent glycan strands of nascent peptidoglycan. We have recently shown that this step can be bypassed by an L,D-transpeptidase, which confers high-level beta-lactam-resistance in Enterococcus faecium. The resistance bypass leads to replacement of D-Ala4-->D-Asx-L-Lys3 cross-links generated by the PBPs by L-Lys3-->D-Asx-L-Lys3 cross-links generated by the L,D-transpeptidase. As the first structure of a member of this new transpeptidase family, we have determined the crystal structure of a fragment of the L,D-transpeptidase from E.faecium (Ldt(fm217)) at 2.4A resolution. Ldt(fm217) consists of two domains, the N-terminal domain, a new mixed alpha-beta fold, and the ErfK_YbiS_YhnG C-terminal domain, a representative of the mainly beta class of protein structures. Residue Cys442 of the C-terminal domain has been proposed to be the catalytic residue implicated in the cleavage of the L-Lys-D-Ala peptide bond. Surface analysis of Ldt(fm217) reveals that residue Cys442 is localized in a buried pocket and is accessible by two paths on different sides of the protein. We propose that the two paths to the catalytic residue Cys442 are the binding sites for the acceptor and donor substrates of the L,D-transpeptidase.     

Predicting kinetic constants of protein-protein interactions based on structural properties

Elucidating kinetic processes of protein–protein interactions (PPI) helps to understand how basic building blocks affect overall behavior of living systems. In this study, we used structure‐based properties to build predictive models for kinetic constants of PPI. A highly diverse PPI dataset, protein–protein kinetic interaction data and structures (PPKIDS), was built. PPKIDS contains 62 PPI with complex structures and kinetic constants measured experimentally. The influence of structural properties on kinetics of PPI was studied using 35 structure‐based features, describing different aspects of complex structures. Linear models for the prediction of kinetic constants were built by fitting with selected subsets of structure‐based features. The models gave correlation coefficients of 0.801, 0.732, and 0.770 for k_off, k_on, and K_d, respectively, in leave‐one‐out cross validations. The predictive models reported here use only protein complex structures as input and can be generally applied in PPI studies as well as systems biology modeling. Our study confirmed that different properties play different roles in the kinetic process of PPI. For example, k_on was affected by overall structural features of complexes, such as the composition of secondary structures, the change of translational and rotational entropy, and the electrostatic interaction; while k_off was determined by interfacial properties, such as number of contacted atom pairs per 100 Ų. This information provides useful hints for PPI design. Proteins 2010;79:720–734. © 2010 Wiley‐Liss, Inc.     

Folding and stability of the isolated Greek key domains of the long-lived human lens proteins gammaD-crystallin and gammaS-crystallin

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric gamma-crystallins and oligomeric beta-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human gammaD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human gammaD-crystallin, as well as the isolated domains of human gammaS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37 degrees C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human gammaD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein.     

Structures of FHOD1-Nesprin1/2 complexes reveal alternate binding modes for the FH3 domain of formins

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