An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding–unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.     

Development of novel lithocholic acid derivatives as vitamin D receptor agonists

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D₃, 1α,25-dihydroxyvitamin D₃ (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells. Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6 bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated derivatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis of the complex of VDR ligand-binding domain (LBD) with 6.     

Normal mode analysis of mouse epidermal growth factor: Characterization of the harmonic motion

Normal mode analysis of mouse epidermal growth factor (mEGF) has been carried out at room temperature. The value of the lowest frequency is 4.1 cm^?1. This mode corresponds to hinge-bending motion between the N-terminal and C-terminal domains of mEGF. This hinge-bending motion corresponds to the “mitten mode.” In this motion, the N-terminal domain is almost rigid. However, the C-terminal domain is found to consist of three rigid segments. Two segments, C33-D46 and G51-R53, are observed moving in the same direction, but L47-W50 moves in the opposite direction. For this mode, the effective Young's modulus turned out to be 1.1 × 10⁹ dyn·^?2. This value is a little larger than that of the mode with the lowest frequency 4.4 cm^?1 of BPTI. The difference may be related to the fraction of residues involved in β-sheets in the molecule. Similar analyses are carried out for other low frequency modes.     

Fluorescence-quenching screening of protein kinase C ligands with an environmentally sensitive fluorophore

A novel fluorescence-quenching screening method for protein kinase C (PKC) ligands was developed utilizing solvatochromic fluorophores. Solvatochromic dyes, highly sensitive to the presence or the absence of competitive ligands in their binding to the C1b domain of PKCδ (δC1b), were combined with a known pharmacophoric moiety of 1,2-diacylglycerol (DAG) lactones, PKC ligands. Addition of δC1b to the fluorescent compounds caused a gradual increase in the fluorescent intensity in proportion to the increase of δC1b. As a competitive ligand was added to the complex of δC1b domain and fluorescent compounds, a gradual decrease in the fluorescent intensity was observed. The relative binding affinities of known ligands were successfully determined by this fluorescent method and corresponded well to the K(i) values measured by a radioisotope method. These results indicate that washing, which is a laborious step in binding evaluations, is not required for this environmentally sensitive fluorophore based system. Screening with the system was performed for 2560 preselected library compounds with possible pharmacophores, and some lead compounds were found. This fluorescence-based method could be applied widely to known ligand-receptor combinations.     

Crystal structural analysis of protein-protein interactions drastically destabilized by a single mutation

The complex of barnase (bn) and barstar (bs), which has been widely studied as a model for quantitative analysis of protein-protein interactions, is significantly destabilized by a single mutation, namely, bs Asp39 --> Ala, which corresponds to a change of 7.7 kcal x mol(-1) in the free energy of binding. However, there has been no structural information available to explain such a drastic destabilization. In the present study, we determined the structure of the mutant complex at 1.58 A resolution by X-ray crystallography. The complex was similar to the wild-type complex in terms of overall and interface structures; however, the hydrogen bond network mediated by water molecules at the interface was significantly different. Several water molecules filled the cavity created by the mutation and consequently caused rearrangement of the hydrated water molecules at the interface. The water molecules were redistributed into a channel-like structure that penetrated into the complex. Furthermore, molecular dynamics simulations showed that the mutation increased the mobility of water molecules at the interface. Since such a drastic change in hydration was not observed in other mutant complexes of bn and bs, the significant destabilization of the interaction may be due to this channel-like structure of hydrated water molecules.     

Structural Analysis of Hen Egg Lysozyme Refolded after Denaturation at Acidic pH

The Protein Journal - Protein structures fluctuate in solution; therefore, proteins have multiple stable structures that are slightly different from each other. In this study, we determined the...     

Requirements for peptidyl-prolyl isomerization activity: a comprehensive mutational analysis of the substrate-binding cavity of FK506-binding protein 12

Peptidyl-prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506-binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate-binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild-type FKBP12. It was found that site-specific interactions by the side chains of amino acid residues constituting the substrate-binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro-containing peptide.     

Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.