Structures and mechanisms of actin ATP hydrolysis.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Raman Spectroscopy of an Atypical C15-E,syn Bilin Chromophore in Cyanobacteriochrome RcaE.

Cyanobacteriochromes (CBCRs) belong to the phytochrome superfamily of photoreceptors, the members of which utilize a linear tetrapyrrole (bilin) as a chromophore. RcaE is a representative member of a green/red-type CBCR subfamily that photoconverts between a green-absorbing dark state and red-absorbing photoproduct (Pr). Our recent crystallographic study showed that the phycocyanobilin (PCB) chromophore of RcaE adopts a unique C15-E,syn configuration in the Pr state, unlike the typical C15-E,anti configuration for the phytochromes and other CBCRs. Here, we measured Raman spectra of the Pr state of RcaE with 1064 nm excitation and explored the structure of PCB and its interacting residues under physiologically relevant aqueous conditions. We also performed measurements of RcaE in D2O as well as the sample reconstituted with the PCB labeled with 15N or with both 13C and 15N. The observed Raman spectra were analyzed by quantum mechanics/molecular mechanics (QM/MM) calculations together with molecular dynamics simulations. The Raman spectra and their isotope effects were well-reproduced by the simulated spectra of fully protonated PCB with the C15-E,syn configuration and allowed us to assign most of the observed bands. The present vibrational analysis of the all syn bilin chromophore using the QM/MM method will advance future studies on CBCRs and the related proteins by vibrational spectroscopy.

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE.

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a "bucket" consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen bonded in the ß-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the "leaky bucket" structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

Crystal structure of human V-1 in the apo form.

V-1, also known as myotrophin, is a 13 kDa ankyrin-repeat protein that binds and inhibits the heterodimeric actin capping protein (CP), which is a key regulator of cytoskeletal actin dynamics. The crystal structure of V-1 in complex with CP revealed that V-1 recognizes CP via residues spanning several ankyrin repeats. Here, the crystal structure of human V-1 is reported in the absence of the specific ligand at 2.3 Šresolution. In the asymmetric unit, the crystal contains two V-1 monomers that exhibit nearly identical structures (Cα r.m.s.d. of 0.47 Å). The overall structures of the two apo V-1 chains are also highly similar to that of CP-bound V-1 (Cα r.m.s.d.s of <0.50 Å), indicating that CP does not induce a large conformational change in V-1. Detailed structural comparisons using the computational program All Atom Motion Tree revealed that CP binding can be accomplished by minor side-chain rearrangements of several residues. These findings are consistent with the known biological role of V-1, in which it globally inhibits CP in the cytoplasm.

A common allosteric mechanism regulates homeostatic inactivation of auxin and gibberellin.

Allosteric regulation is protein activation by effector binding at a site other than the active site. Here, we show via X-ray structural analysis of gibberellin 2-oxidase 3 (GA2ox3), and auxin dioxygenase (DAO), that such a mechanism maintains hormonal homeostasis in plants. Both enzymes form multimers by interacting via GA4 and indole-3-acetic acid (IAA) at their binding interface. Via further functional analyses we reveal that multimerization of these enzymes gradually proceeds with increasing GA4 and IAA concentrations; multimerized enzymes have higher specific activities than monomer forms, a system that should favour the maintenance of homeostasis for these phytohormones. Molecular dynamic analysis suggests a possible mechanism underlying increased GA2ox3 activity by multimerization-GA4 in the interface of oligomerized GA2ox3s may be able to enter the active site with a low energy barrier. In summary, homeostatic systems for maintaining GA and IAA levels, based on a common allosteric mechanism, appear to have developed independently.

High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP.

A high-pressure crystallographic study was conducted on Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ in crystal forms containing both the open and closed conformations of the M20 loop under high-pressure conditions of up to 800 MPa. At pressures between 270 and 500 MPa the crystal form containing the open conformation exhibited a phase transition from P21 to C2. Several structural changes in ecDHFR were observed at high pressure that were also accompanied by structural changes in the NADP+ cofactor and the hydration structure. In the crystal form with the closed conformation the M20 loop moved as the pressure changed, with accompanying conformational changes around the active site, including NADP+ and folate. These movements were consistent with the suggested hypothesis that movement of the M20 loop was necessary for ecDHFR to catalyze the reaction. In the crystal form with the open conformation the nicotinamide ring of the NADP+ cofactor undergoes a large flip as an intermediate step in the reaction, despite being in a crystalline state. Furthermore, observation of the water molecules between Arg57 and folate elucidated an early step in the substrate-binding pathway. These results demonstrate the possibility of using high-pressure protein crystallography as a method to capture high-energy substates or transient structures related to the protein reaction cycle.

Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA.

APOBEC3H (A3H) is a mammal-specific cytidine deaminase that potently restricts the replication of retroviruses. Primate A3Hs are known to exert key selective pressures against the cross-species transmission of primate immunodeficiency viruses from chimpanzees to humans. Despite recent advances, the molecular structures underlying the functional mechanisms of primate A3Hs have not been fully understood. Here, we reveal the 2.20-Å crystal structure of the chimpanzee A3H (cpzA3H) dimer bound to a short double-stranded RNA (dsRNA), which appears to be similar to two recently reported structures of pig-tailed macaque A3H and human A3H. In the structure, the dsRNA-binding interface forms a specialized architecture with unique features. The analysis of the dsRNA nucleotides in the cpzA3H complex revealed the GC-rich palindrome-like sequence preference for dsRNA interaction, which is largely determined by arginine residues in loop 1. In cells, alterations of the cpzA3H residues critical for the dsRNA interaction severely reduce intracellular protein stability due to proteasomal degradation. This suggests that cpzA3H stability is regulated by the dsRNA-mediated dimerization as well as by unknown cellular machinery through proteasomal degradation in cells. Taken together, these findings highlight unique structural features of primate A3Hs that are important to further understand their cellular functions and regulation.

Similar structural stabilities of 3-isopropylmalate dehydrogenases from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 and its atmospheric congener S. oneidensis strain MR-1.

We previously found that the enzymatic activity of 3-isopropylmalate dehydrogenase from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 (SbIPMDH) was pressure-tolerant up to 100 MPa, but that from its atmospheric congener S. oneidensis strain MR-1 (SoIPMDH) was pressure-sensitive. Such characteristics were determined by only one amino acid residue at position 266, serine (SoIPMDH) or alanine (SbIPMDH) [Y. Hamajima et al. Extremophiles 20: 177, 2016]. In this study, we investigated the structural stability of these enzymes. At pH 7.6, SoIPMDH was slightly more stable against hydrostatic pressure than SbIPMDH, contrary to the physiological pressures of their normal environments. Pressure unfolding of these IPMDHs followed a two-state unfolding model between a native dimer and two unfolded monomers, and the dimer structure was pressure-tolerant up to 200 MPa, employing a midpoint pressure of 245.3 ±â€¯0.1 MPa and a volume change of -225 ±â€¯24 mL mol-1 for the most unstable mutant, SbIPMDH A266S. Thus, their pressure-dependent activity did not originate from structural perturbations such as unfolding or dimer dissociation. Conversely, urea-induced unfolding of these IPMDHs followed a three-state unfolding model, including a dimer intermediate. Interestingly, the first transition was strongly pH-dependent but pressure-independent; however, the second transition showed the opposite pattern. Obtained volume changes due to urea-induced unfolding were almost equal for both IPMDHs, approximately +10 and -30 mL mol-1 for intermediate formation and dimer dissociation, respectively. These results indicated that both IPMDHs have similar structural stability, and a pressure-adaptation mechanism was provided for only the enzymatic activity of SbIPMDH.

Protein crystallography beamline BL2S1 at the Aichi synchrotron.

The protein crystallography beamline BL2S1, constructed at one of the 5 T superconducting bending-magnet ports of the Aichi synchrotron, is available to users associated with academic and industrial organizations. The beamline is mainly intended for use in X-ray diffraction measurements of single-crystals of macromolecules such as proteins and nucleic acids. Diffraction measurements for crystals of other materials are also possible, such as inorganic and organic compounds. BL2S1 covers the energy range 7-17 keV (1.8-0.7 Å) with an asymmetric-cut curved single-crystal monochromator [Ge(111) or Ge(220)], and a platinum-coated Si mirror is used for vertical focusing and as a higher-order cutoff filter. The beamline is equipped with a single-axis goniometer, a CCD detector, and an open-flow cryogenic sample cooler. High-pressure protein crystallography with a diamond anvil cell can also be performed using this beamline.

Pressure adaptation of 3-isopropylmalate dehydrogenase from an extremely piezophilic bacterium is attributed to a single amino acid substitution.

3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions. Structural analyses of SoIPMDH suggests that penetration of three water molecules into the cleft around Ser266 under high-pressure conditions could reduce the activity of the wild-type enzyme; however, no water molecule is observed in the Ala266 mutant.

High-pressure protein crystallography of hen egg-white lysozyme.

Crystal structures of hen egg-white lysozyme (HEWL) determined under pressures ranging from ambient pressure to 950 MPa are presented. From 0.1 to 710 MPa, the molecular and internal cavity volumes are monotonically compressed. However, from 710 to 890 MPa the internal cavity volume remains almost constant. Moreover, as the pressure increases to 950 MPa, the tetragonal crystal of HEWL undergoes a phase transition from P43212 to P43. Under high pressure, the crystal structure of the enzyme undergoes several local and global changes accompanied by changes in hydration structure. For example, water molecules penetrate into an internal cavity neighbouring the active site and induce an alternate conformation of one of the catalytic residues, Glu35. These phenomena have not been detected by conventional X-ray crystal structure analysis and might play an important role in the catalytic activity of HEWL.

Pressure effects on the chimeric 3-isopropylmalate dehydrogenases of the deep-sea piezophilic Shewanella benthica and the atmospheric pressure-adapted Shewanella oneidensis.

The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme.

Structural analysis of 3-isopropylmalate dehydrogenase from the obligate piezophile Shewanella benthica DB21MT-2 and the nonpiezophile Shewanella oneidensis MR-1.

Organisms living in deep seas such as the Mariana Trench must be adapted to the extremely high pressure environment. For example, the 3-isopropylmalate dehydrogenase from the obligate piezophile Shewanella benthica DB21MT-2 (SbIPMDH) remains active in extreme conditions under which that from the land bacterium S. oneidensis MR-1 (SoIPMDH) becomes inactivated. In order to unravel the differences between these two IPMDHs, their structures were determined at ~1.5 Å resolution. Comparison of the structures of the two enzymes shows that SbIPMDH is in a more open form and has a larger internal cavity volume than SoIPMDH at atmospheric pressure. This loosely packed structure of SbIPMDH could help it to avoid pressure-induced distortion of the native structure and to remain active at higher pressures than SoIPMDH.

High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase.

Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Šresolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.

A mechanism for single-stranded DNA-binding protein (SSB) displacement from single-stranded DNA upon SSB-RecO interaction.

Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSB-coated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO.