Structural and functional properties of Grb2 SH2 dimer in CD28 binding.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein that plays a critical role in cellular signal transduction. It contains a central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. Binding of Grb2 SH2 to the cytoplasmic region of CD28, phosphorylated Tyr (pY) containing the peptide motif pY-X-N-X, is required for costimulatory signaling in T cells. In this study, we purified the dimer and monomer forms of Grb2 SH2, respectively, and analyzed their structural and functional properties. Size exclusion chromatography analysis showed that both dimer and monomer exist as stable states. Thermal stability analysis using circular dichroism showed that the dimer mostly dissociates into the monomer around 50°C. CD28 binding experiments showed that the affinity of the dimer to the phosphopeptide was about three fold higher than that of the monomer, possibly due to the avidity effect. The present crystal structure analysis of Grb2 SH2 showed two forms; one is monomer at 1.15 Å resolution, which is currently the highest resolution analysis, and another is dimer at 2.00 Å resolution. In the dimer structure, the C-terminal region, comprising residues 123-152, was extended towards the adjacent molecule, in which Trp121 was the hinge residue. The stable dimer purified using size exclusion chromatography would be due to the C-terminal helix "swapping". In cases where a mutation caused Trp121 to be replaced by Ser in Grb2 SH2, this protein still formed dimers, but lost the ability to bind CD28.

Site-specific observation of the conformational change of a protein with 15N-labeled Tyr residues using NMR.

One of the reasons it is difficult to analyze protein structural dynamics at atomic resolution using NMR is the molecular size of the protein. The selective amino acid labeling method is one of the effective methods that can solve this problem. In this study, to determine the site-specific conformational change in 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD), which forms a dimer composed of two 26 kDa subunits, we expressed and purified 15N-Tyr labeled Ps3αHSD and its mutants, and analyzed the conformational change upon NADH binding. Using the Tyr substituted mutants, we first assigned the respective signals of four Tyr residues. In the titration experiments with NADH, the four Tyr signals changed uniquely; changes in chemical shift and signal broadening were observed. The NADH binding affinity, determined from the plots of the 1H and 15N chemical shift changes, was comparable to those reported previously. Together with the crystal structure information for Ps3αHSD in the NADH-free and -bound states, site-specific conformational changes including environmental changes could be deduced.

Binding thermodynamics of metal ions to HIV-1 ribonuclease H domain.

Metal-protein interactions are not necessarily tight in many transient biological processes, such as cellular signaling, enzyme regulation, and molecular recognition. Here, we analyzed the binding thermodynamics and characterized the structural effect of divalent metal ions, i.e. Mn2+, Zn2+, and Mg2+, to the isolated ribonuclease H (RNH) of human immunodeficiency virus (HIV) using isothermal titration calorimetry (ITC) and circular dichroism. The binding thermodynamics of Mg2+ to RNH was determined using competition ITC experiments, and the binding affinity of Mg2+ was found to be about 40- and 400-times lower than those of Mn2+ and of Zn2+, respectively. The structural analysis showed that Mg2+ binding had little effect on the thermal stability of RNH, while Zn2+ and Mn2+ binding increased the stability. The thermodynamic characteristics of RNH metal binding, compared to intact HIV reverse transcriptase, and a possible mechanism of conformational change induced upon metal ion binding, in correlation with the structure-function relationship, are discussed.

Enzymatic hydrolysis of PET: functional roles of three Ca2+ ions bound to a cutinase-like enzyme, Cut190*, and its engineering for improved activity.

Cut190 from Saccharomonospora viridis AHK190 (Cut190) is the only cutinase that exhibits inactive (Ca2+-free) and active (Ca2+-bound) states, although other homologous cutinases always maintain the active states (Ca2+-free and bound). The X-ray crystallography of the S176A mutant of Cut190* (Cut190_S226P/R228S) showed that three Ca2+ ions were bound at sites 1-3 of the mutant. We analyzed the roles of three Ca2+ ions by mutation and concluded that they play different roles in Cut190* for activation (sites 1 and 3) and structural and thermal stabilization (sites 2 and 3). Based on these analyses, we elucidated the mechanism for the conformational change from the Ca2+-free inactive state to the Ca2+-bound active state, proposing the novel Ca2+ effect on structural dynamics of protein. The introduction of a disulfide bond at Asp250 and Glu296 in site 2 remarkably increased the melting temperatures of the mutant enzymes by more than 20-30 °C (while Ca2+-bound) and 4-14 °C (while Ca2+-free), indicating that a disulfide bond mimics the Ca2+ effect. Replacement of surface asparagine and glutamine with aspartic acid, glutamic acid, or histidine increased the melting temperatures. Engineered mutant enzymes were evaluated by an increase in melting temperatures and kinetic values, based on the hydrolysis of poly(butylene succinate-co-adipate) and microfiber polyethylene terephthalate (PET). A combined mutation, Q138A/D250C-E296C/Q123H/N202H, resulted in the highest thermostability, leading to the maximum degradation of PET film (more than 30%; approximately threefold at 70 °C, compared with that of Cut190* at 63 °C).

Structural Dynamics of the PET-Degrading Cutinase-like Enzyme from Saccharomonospora viridis AHK190 in Substrate-Bound States Elucidates the Ca2+-Driven Catalytic Cycle.

A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca2+ binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca2+ binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond is close to the active site and pointing outward from the active site, respectively, for the two complexes. Ca2+ binding induces the pocket to open, enabling the substrate to access the pocket more easily. Molecular dynamics simulations suggest that a post-reaction state in the engaged form presumably exists between the experimentally observed forms, indicating that the substrate would be cleaved in the engaged form and then requires the enzyme to change to the open form to release the product, a process that Ca2+ can greatly accelerate.

DNA-binding induced conformational change of c-Myb R2R3 analyzed using diffracted X-ray tracking.

Previous structural analyses have shown that R2R3, the minimum unit of the DNA-binding domain of the transcriptional factor c-Myb, is largely flexible in solution, and changes to a more rigid structure upon DNA binding. In this study, we evaluated the structural dynamics using the diffracted X-ray tracking method, in correlation with DNA-binding abilities under different salt conditions, and compared them with the previous results. The resultant curve of the mean square angular displacements (MSD) clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry. The results of the MSD curves also indicate that the translational length reduces by approximately half upon DNA binding.

Effects of active site residues of 3α-hydroxysteroid dehydrogenase from pseudomonas sp. b-0831 on its catalysis and cofactor binding.

We overexpressed and purified 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD) and its mutants where the active site residues known as the SYK triad, Ser114, Tyr153, and Lys157, were mutated. Ps3αHSD catalyzes the reaction by using a nucleotide cofactor. The NADH binding affinity of K157A mutant was much lower than that of the wild-type, mainly due to loss of a hydrogen bond. The decreased affinity would result in decreased kcat. Compared to the wild-type, the mutants S114A and Y153F showed higher Km and lower kcat values in both oxidation and reduction reactions. Simultaneous mutation of S114A and Y153F resulted in a significant decrease in kcat relative to the single mutant. These results are supported by the notion that Tyr153 is a catalytic base and Ser114 would be a substitute. Loss of hydrogen bonding with NADH upon the Y153F mutation resulted in increased enthalpy change, partially compensated by increased entropy change.

Structural and thermodynamic characterization of endo-1,3-ß-glucanase: Insights into the substrate recognition mechanism.

Endo-1,3-ß-glucanase from Cellulosimicrobium cellulans is composed of a catalytic domain and a carbohydrate-binding module. We have determined the X-ray crystal structure of the catalytic domain at a high resolution of 1.66Å. The overall fold is a sandwich-like ß-jelly roll architecture like the enzymes in the glycoside hydrolase family 16. The substrate-binding cleft has a length and a width of ~28 and ~15Å, respectively, which is thought to be capable of accommodating at least six glucopyranose units. Laminarihexaose was placed into the substrate-binding cleft, namely at the subsites +2 to -4 from the reducing end, and the complex structure was analyzed using molecular dynamics simulations (MD) and using a rotamer search of the pocket. During the MD simulations, the substrate fluctuated more than the enzyme, where the residues at the subsites toward the non-reducing end fluctuated more than those toward the reducing end. Little conformational change of the protein was observed for the subsites +1 and +2, indicating that the glucose's position could be tightly restricted inside the pocket. Substrate binding experiments using isothermal titration calorimetry showed that the binding affinity of laminaritriose was higher than that of laminaribiose and similar to those of other longer laminarioligosaccharides. Taken together, the substrates mainly bind to the subsites -1 to -3 with the highest affinity, while the part bound to the reducing end would be hydrolyzed.

First observation of metal ion-induced structural fluctuations of α-helical peptides by using diffracted X-ray tracking.

In order to analyze protein structural dynamics, we designed simple model peptides whose structures changed from random-coil to helix-bundle structures by forming stable hydrophobic core in the presence of metal ions. The strategy involved destabilizing a de novo designed three helix-bundle protein by substituting the residues present in its hydrophobic core with histidine and small amino acids. The conformational changes of peptides induced upon binding of Zn2+ to histidine were analyzed using circular dichroism spectroscopy, which revealed peptides, HA and HG, to be good candidates for further analyses. The diffracted X-ray tracking experiments showed that the structural fluctuations of both HA and HG were suppressed upon binding of Zn2+. We succeeded in observing the differences in fluctuations of HA and HG in solution between random-coil like and helix-bundle structures. The metal-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry.

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments.

Tryptophan introduction can change ß-glucan binding ability of the carbohydrate-binding module of endo-1,3-ß-glucanase.

Endo-1,3-ß-glucanase from Cellulosimicrobium cellulans DK-1 has a carbohydrate-binding module (CBM-DK) at the C-terminal side of a catalytic domain. Out of the imperfect tandem α-, ß-, and γ-repeats in CBM-DK, the α-repeat primarily contributes to ß-glucan binding. This unique feature is derived from Trp273 in α-repeat, whose corresponding residues in ß- and γ-repeats are Asp314 and Gly358, respectively. In this study, we generated Trp-switched mutants, W273A/D314W, D270A/W273A/D314W, W273A/G358W, and D270A/W273A/G358W, and analyzed their binding abilities toward laminarioligosaccharides and laminarin. While the binding affinities of D270A/W273A and W273A mutants were either lost or much lower than that of the wild-type, those of Trp-switched mutants recovered, indicating that a Trp introduction in ß- or γ-repeat can substitute the α-repeat by primarily contributing to ß-glucan binding. Thus, we have successfully engineered a CBM-DK that binds to laminarin by a mechanism different from that of the wild-type, but with similar affinity.

Pronounced effect of hapten binding on thermal stability of an anti-(4-hydroxy-3-nitrophenyl)acetyl antibody possessing a glycine residue at position 95 of the heavy chain.

Immune response to T-cell-dependent antigens is highly dynamic; several B-cell clones responsible for antibody production appear alternately during immunization. It was previously shown that at least two-types of antibodies are secreted after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP); one has Tyr and another has Gly at position 95 of the heavy chain (referred to as Tyr95- and Gly95-type). The former appeared at an early stage, while the latter appeared at a late stage, i.e., after secondary immunization, although Fv domains of these antibodies were encoded by same genes of variable heavy and light chains. We examined whether any biophysical properties of antigen-combing sites relate to this shift in B-cell clones by preparing single-chain Fv (scFv). Thermodynamic and kinetic parameters of the interaction of scFv with various haptens are in accordance with those of intact antibodies, indicating that scFvs are appropriate models for the study on structure and function of antibodies. Next, we measured thermal stability of scFvs using differential scanning calorimetry and found that the apparent melting temperature of free Tyr95-type was 64-66°C,while that of Gly95-type was 47-48°C, indicating that the latter was highly unstable. However, Gly95-type greatly gained thermal stability because of hapten binding. We discussed the relationship between thermal stability resulted by hapten binding and dynamism of antibody response during immunization.

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

Structural and binding properties of laminarin revealed by analytical ultracentrifugation and calorimetric analyses.

One of the ß-1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3-ß-glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3-ß-glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3-ß-glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 10(6) M(-1). The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6 kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3-ß-glucanase.

Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.

Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.

Folding thermodynamics of c-Myb DNA-binding domain in correlation with its α-helical contents.

The conformational and thermal stabilities of the minimum functional unit for c-Myb DNA-binding domain, tandem repeat 2 and 3 (R2R3), were analyzed under different pH conditions, ranging from 4.0 to 7.5, using circular dichroism and differential scanning calorimetry. Secondary structure analysis showed that the solution pH largely affects the conformational stability of the protein domain. Of all conditions analyzed, the α-helical content was maximal at pH 6.5, and the thermal stability was highest at pH 5.0. Thermodynamic parameters for thermal unfolding of R2R3 were determined using differential scanning calorimetry, and the origin of folding thermodynamics at the different pHs and its correlation with the α-helical content were further analyzed. It should be noted that the α-helical content correlates well with the enthalpy change in the pH range from 4.5 to 7.5, suggesting that the strength of hydrogen bonds and salt bridges needed for maintenance of helical structure is related to enthalpy in the native state. Under physiological pH conditions, c-Myb R2R3 exists in the enthalpically unstable but entropically stable state. Due to loss of rigid structure and high stability, the protein can now obtain structural flexibility, befitting its function.

Functional conformer of c-Myb DNA-binding domain revealed by variable temperature studies.

The conformational fluctuation in the minimum DNA-binding domain of c-Myb, repeats 2 and 3 (R2R3), was studied under closely physiological conditions. A global unfolding transition, involving both the main chain and the side chains, was found to take place at the approximate temperature range 30-70 °C, with a transition temperature of approximately 50 °C. In addition, the observation of simultaneous shift change and broadening of NMR signals in both (1)H one-dimensional and (15)N/(1)H two-dimensional NMR spectra indicated the occurrence of locally fluctuating state at physiological temperature. In the wild-type protein containing a cavity in R2, the local fluctuation of R2 is more prominent than that of R3, whereas it is suppressed in the cavity-filled mutant, V103L. This indicates that the cavity in R2 contributes significantly to the conformational instability and the transition into the locally fluctuating state. For the wild-type R2R3 protein, the more dynamic conformer is estimated to be present to some extent at 37 °C and is likely beneficial for its biological function: DNA-binding. This result is in agreement with the concept of an excited-state conformer that exists in equilibrium with the dominant ground-state conformer and acts as the functional conformer of the protein. From the findings of the present study, it appears that the tandem repeats of two small domains with no disulfide bonds and with a destabilizing cavity function as the evolutionary strategy of the wide-type c-Myb DNA-binding domain to produce an appropriate fraction of the locally fluctuating state at 37 °C, which is more amenable to DNA-binding. Database: Chemical shifts and peak lists have been deposited in the Biological Magnetic Resonance Bank under entries 11584 and 11585.

Structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions.

We extracted collagen from moon jellyfish under neutral pH conditions and analyzed its amino acid composition, secondary structure, and thermal stability. The content of hydroxyproline was 4.3%, which is lower than that of other collagens. Secondary structure analysis using circular dichroism (CD) showed a typical collagen helix. The thermal stability of this collagen at pH 3.0 was lower than those from fish scale and pig skin, which also correlates closely with jellyfish collagen having lower hydroxyproline content. Because the solubility of jellyfish collagen used in this study at neutral pH was quite high, it was possible to analyze its structural and physical properties under physiological conditions. Thermodynamic analysis using CD and differential scanning calorimetry showed that the thermal stability at pH 7.5 was higher than at pH 3.0, possibly due to electrostatic interactions. During the process of unfolding, fibrillation would occur only at neutral pH.

Crystal structures of hereditary vitamin D-resistant rickets-associated vitamin D receptor mutants R270L and W282R bound to 1,25-dihydroxyvitamin D3 and synthetic ligands.

The vitamin D receptor (VDR), a member of the nuclear receptor superfamily, functions as a ligand-dependent transcription factor for various genes. Hereditary vitamin D-resistant rickets (HVDRR), an autosomal recessive disease, is caused by mutations in the VDR. In particular, the missense mutations R274L and W286R in the ligand-binding domain of the VDR can severely reduce or even eliminate natural hormone responsiveness. Here, we report a crystal structure analysis of the R270L and W282R mutants of rat VDR (human R274L and W286R, respectively) in complex with the natural hormone and synthetic ligands. We also studied the folding properties of the mutant proteins by using circular dichroism spectra. Our study indicates that these mutations result in only local structural modifications. We discuss why these mutations disrupt the VDR function and provide clues to develop effective ligands for the treatment of HVDRR.

Thermodynamic effects of multiple protein conformations on stability and DNA binding.

The side-chain conformations of amino acids in the hydrophobic core are important for protein folding and function. A previous NMR study has shown that a mutant protein of transcriptional activator c-Myb, I155L/I181L R3, has multiple conformations and increased fluctuation in comparison with the wild type. To elucidate the quantitative correlation of structural fluctuation with stability and function, we analyzed the thermodynamic effects of I155L and I181L mutations, using R2R3 that encompasses the minimum specific DNA-binding region. Circular dichroism and differential scanning calorimetry measurements showed that the mutation of I155L had little effect on stability, while the I181L mutation significantly destabilized the protein. It is noteworthy that the decreased stability resulting from the I181L mutation was mainly due to decreased enthalpy change, which is partially compensated by decreased entropy change. Isothermal titration calorimetry measurements showed that the specific DNA-binding affinity was decreased owing to the I181L mutation, which was due to decreased binding entropy change. Entropy in the folded state, which corresponds to the DNA-free state, increases due to the I181L mutation because of the increased conformational fluctuation observed in I155L/I181L mutant of R2R3 by CLEANEX-PM NMR analysis, which in turn results in decreased folding entropy and DNA-binding entropy changes.