Hydrogen exchange and ligand binding: ligand-dependent and ligand-independent protection in the Src SH3 domain

Amide hydrogen-deuterium exchange has proven to be a powerful tool for detecting and characterizing high-energy conformations in protein ensembles. Since interactions with ligands can modulate these high-energy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can complicate such studies. Here, we examine hydrogen exchange rates in a simple model system, the c-Src SH3 domain interacting with a short peptide ligand. Addition of ligand slows the rates of hydrogen exchange at nearly every amide for which we can obtain data. Careful analysis, however, reveals that this slowing is due primarily to a reduction in the population of free protein in the system, and not to any specific property of the complex. We present a method to separate the contributions of free and bound protein to the exchange kinetics that has allowed us to identify the subset of amides where exchange arises directly from the complex. These results demonstrate that the slowing of hydrogen exchange induced by ligand interactions should be interpreted with caution, and more extensive experiments are required to correlate changes in hydrogen exchange with changes in structure or internal dynamics.     

Role of amino acid residues in left-handed helical conformation for the conformational stability of a protein

Our previous study of six non-Gly to Gly/Ala mutant human lysozymes in a left-handed helical region showed that only one non-Gly residue at a rigid site had unfavorable strain energy as compared with Gly at the same position (Takano et al., Proteins 2001; 44:233-243). To further examine the role of left-handed residues in the conformational stability of a protein, we constructed ten Gly to Ala mutant human lysozymes. Most Gly residues in human lysozyme are located in the left-handed helix region. The thermodynamic parameters for denaturation and crystal structures were determined by differential scanning calorimetry and X-ray analysis, respectively. The difference in denaturation Gibbs energy (DeltaDeltaG) for the ten Gly to Ala mutants ranged from + 1.9 to -7.5 kJ/mol, indicating that the effect of the mutation depends on the environment of the residue. We confirm that Gly in a left-handed region is more favorable at rigid sites than non-Gly, but there is little difference in energetic cost between Gly and non-Gly at flexible sites. The present results indicate that dihedral angles in the backbone conformation and also the flexibility at the position should be considered for analyses of protein stability, and protein structural determination, prediction, and design.     

Role of non-glycine residues in left-handed helical conformation for the conformational stability of human lysozyme.

To understand the role of non-Gly residues in the left-handed helical conformation for the conformational stability of a protein, the non-Gly to Gly and Ala mutations at six left-handed residues (R21, Y38, R50, Q58, H78, and N118) of the human lysozyme were examined. The thermodynamic parameters for denaturation were determined using a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. If a left-handed non-Gly had an unfavorable steric interaction between the side-chain Cbeta and backbone, the Gly mutation would be expected to stabilize more than the Ala mutation at the same position. For the mutant human lysozymes, however, there were few differences in the denaturation Gibbs energy (DeltaG) between the Gly and Ala mutants, except for the substitution at position 58. Analysis of the changes in stability (DeltaDeltaG) based on the structures of the wild-type and mutant proteins showed that the experimental DeltaDeltaG value of Q58G was approximately 7 kJ/mol higher than the estimated value without consideration of any local steric interaction. These results indicate that only Q58G increased the stability by elimination of local constraints. The residue 58 is located at the most rigid position in the left-handed non-Gly residues and is involved in its enzymatic function. It can be concluded that the left-handed non-Gly residues do not always have unfavorable strain energies as compared with Gly at the same position.     

Distal heme pocket regulation of ligand binding and stability in soybean leghemoglobin

Leghemoglobins facilitate diffusion of oxygen through root tissue to a bacterial terminal oxidase in much the same way that myoglobin transports oxygen from blood to muscle cell mitochondria. Leghemoglobin serves an additional role as an oxygen scavenger to prevent inhibition of nitrogen fixation. For this purpose, the oxygen affinity of soybean leghemoglobin is 20-fold greater than myoglobin, resulting from an 8-fold faster association rate constant combined with a 3-fold slower dissociation rate constant. Although the biochemical mechanism used by myoglobin to bind oxygen has been described in elegant detail, an explanation for the difference in affinity between these two structurally similar proteins is not obvious. The present work demonstrates that, despite their similar structures, leghemoglobin uses methods different from myoglobin to regulate ligand affinity. Oxygen and carbon monoxide binding to a comprehensive set of leghemoglobin distal heme pocket mutant proteins in comparison to their myoglobin counterparts has revealed some of these mechanisms. The "distal histidine" provides a crucial hydrogen bond to stabilize oxygen in myoglobin but has little effect on bound oxygen in leghemoglobin and is retained mainly for reasons of protein stability and prevention of heme loss. Furthermore, soybean leghemoglobin uses an unusual combination of HisE7 and TyrB10 to sustain a weak stabilizing interaction with bound oxygen. Thus, the leghemoglobin distal heme pocket provides a much lower barrier to oxygen association than occurs in myoglobin and oxygen dissociation is regulated from the proximal heme pocket.     

A novel mechanism of ligand binding and release in the odorant binding protein 20 from the malaria mosquito Anopheles gambiae

Anopheles gambiae mosquitoes that transmit malaria are attracted to humans by the odor molecules that emanate from skin and sweat. Odorant binding proteins (OBPs) are the first component of the olfactory apparatus to interact with odorant molecules, and so present potential targets for preventing transmission of malaria by disrupting the normal olfactory responses of the insect. AgamOBP20 is one of a limited subset of OBPs that it is preferentially expressed in female mosquitoes and its expression is regulated by blood feeding and by the day/night light cycles that correlate with blood‐feeding behavior. Analysis of AgamOBP20 in solution reveals that the apo‐protein exhibits significant conformational heterogeneity but the binding of odorant molecules results in a significant conformational change, which is accompanied by a reduction in the conformational flexibility present in the protein. Crystal structures of the free and bound states reveal a novel pathway for entrance and exit of odorant molecules into the central‐binding pocket, and that the conformational changes associated with ligand binding are a result of rigid body domain motions in α‐helices 1, 4, and 5, which act as lids to the binding pocket. These structures provide new insights into the specific residues involved in the conformational adaptation to different odorants and have important implications in the selection and development of reagents targeted at disrupting normal OBP function.     

Role of C-terminal region of Staphylococcal nuclease for foldability,stability, and activity

The role of the C-terminal region of Staphylococcal nuclease (SNase) was examined by deletion mutation. Deletions up to eight residues do not affect the structure and function. The structure and enzymatic activity were partially lost by deleting Ser141-Asn149 (Delta141-149), and deletion of Trp140-Asn149 (Delta140-149) resulted in further loss of structure and activity. A 13-residue deletion showed the same effect as the 10-residue deletion. Both Ser141Gln and Ser141Ala mutations for an eight-residue deletion mutant did not alter properties as well as Ser141A1a for full-length SNase. In contrast, Trp140Ala mutation for Delta141-149 shows the same effect as the deletion of Trp140. Trp140Ala mutation for full-length SNase causes the loss of native structure. These observations indicate the significance of the 140th and the 141st residues. The side-chain of the 140th residue is required to be tryptophan; however, the backbone of the 141st residue is solely critical for foldability, but the side-chain information is not crucial. All of the mutants that take a non-native conformation show enzymatic activity and inhibitor-induced folding, suggesting that foldability is required for the activity.     

Sequence,biophysical, and structural analyses of the PstS lipoprotein (BB0215) from Borrelia burgdorferi reveal a likely binding component of an ABC‐type phosphate transporter

The Lyme disease agent Borrelia burgdorferi, which is transmitted via a tick vector, is dependent on its tick and mammalian hosts for a number of essential nutrients. Like other bacterial diderms, it must transport these biochemicals from the extracellular milieu across two membranes, ultimately to the B. burgdorferi cytoplasm. In the current study, we established that a gene cluster comprising genes bb0215 through bb0218 is cotranscribed and is therefore an operon. Sequence analysis of these proteins suggested that they are the components of an ABC‐type transporter responsible for translocating phosphate anions from the B. burgdorferi periplasm to the cytoplasm. Biophysical experiments established that the putative ligand‐binding protein of this system, BbPstS (BB0215), binds to phosphate in solution. We determined the high‐resolution (1.3 Å) crystal structure of the protein in the absence of phosphate, revealing that the protein's fold is similar to other phosphate‐binding proteins, and residues that are implicated in phosphate binding in other such proteins are conserved in BbPstS. Taken together, the gene products of bb0215‐0218 function as a phosphate transporter for B. burgdorferi.     

Insights into nonspecific binding of homeodomains from a structure of MATalpha2 bound to DNA

The 2.1-A resolution crystal structure of the MATalpha2 homeodomain bound to DNA reveals the unexpected presence of two nonspecifically bound alpha2 homeodomains, in addition to the two alpha2 homeodomains bound to canonical alpha2 binding sites. One of the extra homeodomains makes few base-specific contacts, while the other extra homeodomain binds to DNA in a previously unobserved manner. This unusually bound homeodomain is rotated on the DNA, making possible major groove contacts by side-chains that normally do not contact the DNA. This alternate docking may represent one way in which homeodomains sample nonspecific DNA sequences.     

Cooperative interactions and a non-native buried Trp in the unfolded state of an SH3 domain

The presence of residual structure in the unfolded state of the N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) has been investigated using far- and near-UV circular dichroism (CD), fluorescence, and NMR spectroscopy. The unfolded (U(exch)) state of the drkN SH3 domain is significantly populated and exists in equilibrium with the folded (F(exch)) state under non-denaturing conditions near physiological pH. Denaturation experiments have been performed on the drkN SH3 domain in order to monitor the change in ellipticity, fluorescence intensity, and chemical shift between the U(exch) state and chemically or thermally denatured states. Differences between the unfolded and chemically or thermally denatured states highlight specific areas of residual structure in the unfolded state that are cooperatively disrupted upon denaturation. Results provide evidence for cooperative interactions in the unfolded state involving residues of the central beta-sheet, particularly the beta4 strand. Denaturation as well as hydrogen-exchange experiments demonstrate a non-native burial of the Trp ring within this "cooperative" core of the unfolded state. These findings support the presence of non-native hydrophobic clusters, organised by Trp rings, within disordered states.     

Intramolecular binding of a proximal PPII helix to an SH3 domain in the fusion protein SH3Hck: PPIIhGAP

SH3 domains are a conserved feature of many nonreceptor protein tyrosine kinases, such as Hck, and often function in substrate recruitment and regulation of kinase activity. SH3 domains modulate kinase activity by binding to polyproline helices (PP_II helix) either intramolecularly or in target proteins. The preponderance of bimolecular and distal interactions between SH3 domains and PP_II helices led us to investigate whether proximal placement of a PP_II helix relative to an SH3 domain would result in tight, intramolecular binding. We have fused the PP_II helix region of human GAP to the C-terminus of Hck SH3 and expressed the recombinant fusion protein in Eschericheria coli. The fusion protein, SH3_Hck: PPII_hGAP, folded spontaneously into a structure in which the PP_II helix was bound intramolecularly to the hydrophobic crevice of the SH3 domain. The SH3_Hck: PPII_hGAP fusion protein is useful for investigating SH3: PP_II helix interactions, for studying concepts in protein folding and design, and may represent a protein structural motif that is widely distributed in nature.     

Thermal stabilization of the protozoan Entamoeba histolytica alcohol dehydrogenase by a single proline substitution

Analysis of the three-dimensional structures of two closely related thermophilic and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Entamoeba histolytica (EhADH1) and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro275) at the center of the dimerization interface might be crucial for maintaining the thermal stability of TbADH. To assess the contribution of Pro275 to the thermal stability of the ADHs, we applied site-directed mutagenesis to replace Asp275 of EhADH1 with Pro (D275P-EhADH1) and conversely Pro275 of TbADH with Asp (P275D-TbADH). The results indicate that replacing Asp275 with Pro significantly enhances the thermal stability of EhADH1 (DeltaT(1/2) 相似文献   

Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.     

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same betaalphabetabetaalphabeta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8 degrees C and an unfolding free energy, DeltaG(U-F)H2O, at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.     

Structures of dimeric dihydrodiol dehydrogenase apoenzyme and inhibitor complex: probing the subunit interface with site-directed mutagenesis

Dimeric dihydrodiol dehydrogenase (DD) catalyses the nicotinamide adenine dinucleotide phosphate (NADP+)-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons to their corresponding catechols. This is the first report of the crystal structure of the dimeric enzyme determined at 2.0 A resolution. The tertiary structure is formed by a classical dinucleotide binding fold comprising of two betaalphabetaalphabeta motifs at the N-terminus and an eight-stranded, predominantly antiparallel beta-sheet at the C-terminus. The active-site of DD, occupied either by a glycerol molecule or the inhibitor 4-hydroxyacetophenone, is located in the C-terminal domain of the protein and maintained by a number of residues including Lys97, Trp125, Phe154, Leu158, Val161, Asp176, Leu177, Tyr180, Trp254, Phe279, and Asp280. The dimer interface is stabilized by a large number of intermolecular contacts mediated by the beta-sheet of each monomer, which includes an intricate hydrogen bonding network maintained in principal by Arg148 and Arg202. Site-directed mutagenesis has demonstrated that the intact dimer is not essential for catalytic activity. The similarity between the quaternary structures of mammalian DD and glucose-fructose oxidoreductase isolated from the prokaryotic organism Zymomonas mobilis suggests that both enzymes are members of a unique family of oligomeric proteins and may share a common ancestral gene.     

Mapping the lifetimes of local opening events in a native state protein.

The rate constants for the processes that lead to local opening and closing of the structures around hydrogen bonds in native proteins have been determined for most of the secondary structure hydrogen bonds in the four-helix protein acyl coenzyme A binding protein. In an analysis that combines these results with the energies of activation of the opening processes and the stability of the local structures, three groups of residues in the protein structure have been identified. In one group, the structures around the hydrogen bonds have frequent openings, every 600 to 1,500 s, and long lifetimes in the open state, around 1 s. In another group of local structures, the local opening is a very rare event that takes place only every 15 to 60 h. For these the lifetime in the open state is also around 1 s. The majority of local structures have lifetimes between 2,000 and 20,000 s and relatively short lifetimes of the open state in the range between 30 and 400 ms. Mapping of these groups of amides to the tertiary structure shows that the openings of the local structures are not cooperative at native conditions, and they rarely if ever lead to global unfolding. The results suggest a mechanism of hydrogen exchange by progressive local openings.     

Stability and interactions of the amino-terminal domain of ClpB from Escherichia coli

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates aggregated proteins. The sequence of ClpB contains two ATP-binding regions that are enclosed between the N- and C-terminal extensions. Whereas it has been found that the N-terminal region of ClpB is essential for the chaperone activity, the structure of this region is not known, and its biochemical properties have not been studied. We expressed and purified the N-terminal fragment of ClpB (residues 1-147). Circular dichroism of the isolated N-terminal region showed a high content of alpha-helical structure. Differential scanning calorimetry showed that the N-terminal region of ClpB is thermodynamically stable and contains a single folding domain. The N-terminal domain is monomeric, as determined by gel-filtration chromatography, and the elution profile of the N-terminal domain does not change in the presence of the N-terminally truncated ClpB (ClpBDeltaN). This indicates that the N-terminal domain does not form strong contacts with ClpBDeltaN. Consistently, addition of the separated N-terminal domain does not reverse an inhibition of ATPase activity of ClpBDeltaN in the presence of casein. As shown by ELISA measurements, full-length ClpB and ClpBDeltaN bind protein substrates (casein, inactivated luciferase) with similar affinity. We also found that the isolated N-terminal domain of ClpB interacts with heat-inactivated luciferase. Taken together, our results indicate that the N-terminal fragment of ClpB forms a distinct domain that is not strongly associated with the ClpB core and is not required for ClpB interactions with other proteins, but may be involved in recognition of protein substrates.