Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of ...unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320
μs after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at ∼22
ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations.
The structure of a histidine kinase (ThkA) complexed with a response regulator (TrrA) in the two-component regulatory system from hyperthermophile Thermotoga maritima was determined by a combination ...of X-ray crystallography at a resolution of 4.2 Å and small-angle X-ray scattering (SAXS). The boundary of the three component domains (PAS-sensor, dimerization and catalytic domains) of ThkA and the bound TrrA molecule were unambiguously assigned in the electron density map at 4.2 Å resolution. ThkA forms a dimer with crystallographic 2-fold symmetry and two monomeric TrrAs bind to the ThkA dimer. SAXS experiments also confirmed this association state in solution and specific binding between ThkA and TrrA (Kd=8.2×10-11 M-2). The association interface between ThkA and TrrA contains the phosphotransfer His residue in the ThkA, indicative of an efficient receipt of the phosphoryl group. One Per-Arnt-Sim (PAS) domain does not interact with the other PAS domain, but with the catalytic domain of the same polypeptide chain and with one TrrA molecule. Observed inter-domain and inter-molecular interactions reveal a definite pathway of signal transduction in the kinase/regulator complex. In addition, we propose a responsible role of TrrA for the feedback regulation of sensing and/or kinase activities of ThkA.
KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase) activity, and its C-terminal ...domain has autokinase and autophosphatase activities targeting own S431 and T432. The coordination of these multiple biochemical activities is the molecular basis for robust circadian rhythmicity. Therefore, much effort has been devoted to elucidating the cooperative relationship between the two domains. However, structural and functional relationships between the two domains remain unclear especially with respect to the dawn phase, at which KaiC relieves its nocturnal history through autodephosphorylation. In this study, we attempted to design a double mutation of S431 and T432 that can capture KaiC as a fully dephosphorylated form with minimal impacts on its structure and function, and investigated the cooperative relationship between the two domains in the night to morning phases from many perspectives. The results revealed that both domains cooperate at the dawn phase through salt bridges formed between the domains, thereby non-locally co-activating two events, ATPase de-inhibition and S431 dephosphorylation. Our further analysis using existing crystal structures of KaiC suggests that the states of both domains are not always in one-to-one correspondence at every phase of the circadian cycle, and their coupling is affected by the interactions with KaiA or adjacent subunits within a KaiC hexamer.
In this review, a longstanding question in chronobiology is revisited: How is the circadian period of biological clocks determined? The origins of the slowness and periodicity should be best studied ...by the interdisciplinary approach employing biophysics and structural biology.
The helix formation dynamics of poly-l-glutamic acids (PGAs) were observed by the microsecond-resolved Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. The helix ...formation of 34-residue PGA from random coil at pH (or pD for FTIR) 8.0 was initiated by a pH jump to 4.9 using the rapid solution mixer whose mixing dead time is 50 μs. The amide I‘ line in the time-resolved FTIR spectra exhibited the fast (<100 μs) increase of the total helical content. The time-resolved CD spectra of the same process also showed the fast (<150 μs) formation of short helical segments (5 ± 1 residues), which was followed by the slower (<1 ms) elongation of the short helices to longer helices (>10 residues). Similar dynamics were observed for the same pH jump of ∼190-residue PGA, although there were additional steps that made the helix formation of ∼190-residue PGA more complex. The observed multistep helix formation is likely caused by the strong hydrogen-bonding interactions between the protonated side chains of PGAs.
CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation ...mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration (
R
g) and the second virial coefficient (
A
2) were 25.3(±0.5)
Å and −0.39(±0.25)×10
−4
ml
mol
g
−2, respectively. CO binding caused a slight increase in
R
g (by 0.5
Å) and a marked decrease in
A
2 (by 5.09×10
−4
ml
mol
g
−2). The observed decrease in
A
2 points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of
R
g rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental
R
g and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8° around the axis of 2-fold symmetry.
P5, also known as PDIA6, is a PDI family member involved in the ER quality control. Here, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like ...domain. Unlike conventional leucine zipper motifs with leucine residues every two helical turns on ∼30-residue parallel α helices, this adhesive motif includes periodic repeats of leucine/valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α helices. The P5 dimerization interface is further stabilized by several reciprocal salt bridges and C-capping interactions between protomers. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as aberrant proteins that induce the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via intermolecular disulfide bond reduction and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and function of P5.
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•P5 dimerizes via a unique leucine-valine adhesive motif in the first Trx-like domain•The adhesive motif is radically different from conventional coiled-coil motifs•The dimeric structure serves to promote P5-mediated inactivation of IRE1α•Ca2+-binding regions and Ca2+-dependent functional regulation were clarified for P5
Using SAXS and X-ray crystallography, Okumura et al. determined the structure of a protein disulfide isomerase P5, in which the N-terminal thioredoxin-like domain dimerizes via a unique leucine-valine adhesive motif. A monomeric P5 mutant displayed structural instability and compromised functions, indicating the structural and functional essentiality of this motif.
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