Satomi Inaba

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.

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.

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 1H one‐dimensional and 15N/1H 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.

Thermodynamic effects of a linker region between two repeats of a protein, c-Myb R2R3, on its stability and structural dynamics

Protein folding thermodynamics can determine the contribution of protein fluctuation, which is difficult to detect but critical for protein function. We have analyzed the structural dynamic properties of the DNA-binding domain of a transcriptional activator, c-Myb R2R3. Earlier reports state that a substitution at the linker between R2 and R3 resulted in a significant loss of affinity toward the cognate DNA, mainly due to increased entropy in the DNA-unbound state. In this study, we analyzed the effects of the linker on folding stability and found that mutation of Pro140 to Gly or Ala resulted in decreased stability, due to an unfavorable enthalpy change that was only partially compensated by a favorable entropy change. Considering that the mutation would increase protein fluctuations in both the folded and unfolded states, we assume that the change in the folded state would be larger than in the unfolded state. This is perhaps due to the additional intramolecular interactions that only occur in the folded state. The increase in protein fluctuation in the folded state post-mutation was correlated with protein function, as reported previously.

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.

Effect of a salt-bridge between inter-repeats on the 3D structure of the c-Myb DNA-binding domain revealed by thermodynamic analysis

Structure folding dictates protein functions. Recent advances in structural analysis such as NMR and X-ray crystallography enabled to determine protein structures at high resolution, which helps understanding of protein folding and recognition mechanisms. Additionally, solution thermodynamic analysis provides useful information to visualize the real behavior of proteins because they largely fluctuate in solution. In this study, we analyzed the effect of a putative salt-bridge between His-137 and Asp-178 in the c-Myb DNA-binding domain on the thermodynamics of its folding and motion. The minimum unit for specific DNA-binding of c-Myb consists of the two repeats, R2 and R3. The residues, His-137 and Asp-178, are located in R2 and R3, respectively, and are spatial proximity, possibly forming a salt-bridge as revealed by a previous crystal structure analysis. D178N mutation caused slight changes in the tertiary structure of R2R3. The thermal stability of the D178N mutant in the absence of DNA was much lower than that of wild-type R2R3. The decrease in stability was due to unfavorable enthalpy change, partially compensated by the favorable entropy change. The largely decreased enthalpy change indicated that the disruption of the salt-bridge weakens the overall intramolecular interactions in the folded state. Additionally, the increased entropy change indicated that the dynamics of the folded state increase upon mutation. In contrast, the D178N mutation slightly affected the thermal stability of the DNA-bound state, indicating that the salt-bridge is required for proper folding of R2R3 in the DNA-free state.

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 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. CD and DSC analyses for the thermal denaturation of jellyfish collagen at pH 7.5 and 3.0 with its schematic

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

Endo-1,3-β-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 β-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 glucoses 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.