Mika Hirose

Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell

We investigated the effect of protein disulfide isomerase (PDI) on in vivo protein folding of human lysozyme (h‐LZM) in a specially constructed yeast coexpression system. Coexpression with PDI increased the amounts of intracellular h‐LZM with the native conformation, leading to an increase in h‐LZM secretion. The results indicated that PDI is a real catalyst of protein folding in the cell. The secretion of h‐LZM increased even when both active sites of PDI were disrupted, suggesting that the effect of PDI resulted from a function other than the formation of disulfide bonds. This is the first finding that PDI without isomerase activity accelerates protein folding in vivo.

OAZ-t/OAZ3 Is Essential for Rigid Connection of Sperm Tails to Heads in Mouse

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. Although considerable amounts of polyamines are synthesized and stored in the testes, their roles remain unknown. Ornithine decarboxylase antizymes (OAZs) control the intracellular concentration of polyamines in a feedback manner. OAZ1 and OAZ2 are expressed ubiquitously, whereas OAZ-t/OAZ3 is expressed specifically in germline cells during spermiogenesis. OAZ-t reportedly binds to ornithine decarboxylase (ODC) and inactivates ODC activity. In a prior study, polyamines were capable of inducing a frameshift at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t. To investigate the physiological role of OAZ-t, we generated OAZ-t–disrupted mutant mice. Homozygous OAZ-t mutant males were infertile, although the polyamine concentrations of epididymides and testes were normal in these mice, and females were fertile. Sperm were successfully recovered from the epididymides of the mutant mice, but the heads and tails of the sperm cells were easily separated in culture medium during incubation. Results indicated that OAZ-t is essential for the formation of a rigid junction between the head and tail during spermatogenesis. The detached tails and heads were alive, and most of the headless tails showed straight forward movement. Although the tailless sperm failed to acrosome-react, the heads were capable of fertilizing eggs via intracytoplasmic sperm injection. OAZ-t likely plays a key role in haploid germ cell differentiation via the local concentration of polyamines.

Growth of Protein Crystals in Hydrogels Prevents Osmotic Shock

High-throughput protein X-ray crystallography offers a significant opportunity to facilitate drug discovery. The most reliable approach is to determine the three-dimensional structure of the protein-ligand complex by soaking the ligand in apo crystals. However, protein apo crystals produced by conventional crystallization in a solution are fatally damaged by osmotic shock during soaking. To overcome this difficulty, we present a novel technique for growing protein crystals in a high-concentration hydrogel that is completely gellified and exhibits high strength. This technique allowed us essentially to increase the mechanical stability of the crystals, preventing serious damage to the crystals caused by osmotic shock. Thus, this method may accelerate structure-based drug discoveries.

Structure Basis for the Regulation of Glyceraldehyde-3-Phosphate Dehydrogenase Activity via the Intrinsically Disordered Protein CP12

The reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex.

Water-Mediated Recognition of Simple Alkyl Chains by Heart-Type Fatty-Acid-Binding Protein†

Long-chain fatty acids (FAs) with low water solubility require fatty-acid-binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub-Angstrom X-ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart-type FABP (FABP3) preferentially incorporates a U-shaped FA of C10–C18 using a lipid-compatible water cluster, and excludes longer FAs using a chain-length-limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.

Promotion of Crystal Nucleation of Protein by Semi-Solid Agarose Gel

We investigate the nucleation rate of protein crystals in the presence of agarose gel at concentrations between 0.0 and 2.0% (w/v). It was found that agarose gel promoted the number of protein crystals up to a concentration of 0.4% (w/v), and gel at concentrations between 0.4 and 0.8% (w/v) decreased the nucleation rate. However, the number of crystals increased again in the presence of agarose by more than 1.0% (w/v), demonstrating high gel strength and a semi-solid gel. We propose that a crystallization method using semi-solid agarose is powerful for the screening of crystallization conditions.

Meichroacidin Containing the Membrane Occupation and Recognition Nexus Motif Is Essential for Spermatozoa Morphogenesis

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.

Protein Crystallization in Agarose Gel with High Strength: Developing an Automated System for Protein Crystallographic Processes

Agarose gel media reduce convection and prevent crystal sedimentation, resulting in the production of high-quality protein crystals. However, crystallographers have only tested agarose gel at concentrations between 0.0 and 0.6% (w/v), where it exhibits low gel strength. The effect of agarose gel on protein structures remains to be elucidated, because only a few structural studies have been performed using gel-grown protein crystals. Here, we crystallize thaumatin and elastase using a variety of crystallization methods in 2.0% (w/v) agarose gels, which are completely gellified and have sufficiently high-strength. This new crystallization approach using semi-solid agarose gels is compatible with several conventional crystallization techniques. A comparison of structures crystallized in non-gelled solution and those crystallized in 2.0% (w/v) agarose gels indicates that the crystal structures were not affected by the high-concentration agarose gels. This technique offers the practical advantages of efficient protection by the semi-solid gel media surrounding the protein crystals, allowing them to be handled and transported without affecting any later crystallographic analysis, and thereby providing an automated system for crystal capturing and mounting.

Crystal structure of FtsA from Staphylococcus aureus

The bacterial cell‐division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA and FtsZ interact during membrane division remains obscure. We have solved 2.2 Å resolution crystal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer units are twisted, in contrast to the straight filament of FtsA from Thermotoga maritima, and the half of S12–S13 hairpin regions are disordered. We confirmed that SaFtsZ and SaFtsA associate in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA.

Approach for growth of high-quality and large protein crystals

Three crystallization methods, including crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study, crystallization has been further evaluated in the presence of a semi-solid agarose gel by crystallizing additional proteins. A novel crystallization method combining TSSG and the large-scale hanging-drop method has also been developed.