Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP.

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane ß sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.

Genetic diversification of the Kanehira bitterling Acheilognathus rhombeus inferred from mitochondrial DNA, with comments on the phylogenetic relationship with its sister species Acheilognathus barbatulus.

The Kanehira bitterling, Acheilognathus rhombeus, is a freshwater fish, discontinuously distributed in western Japan and the Korean Peninsula. Unusually among bitterling it is an autumn-spawning species and shows developmental diapause. Consequently, the characterization of its evolutionary history is significant not only in the context of the fish assemblage of East Asia, but also for understanding life-history evolution. This study aimed to investigate the phylogeography of A. rhombeus and its sister species Acheilognathus barbatulus, distributed in China, using a mitochondrial analysis of the ND1 gene from 311 samples collected from 50 localities in Japan and continental Asia. Phylogenetic analysis revealed that A. barbatulus is included in A. rhombeus and genetically closer to Japanese A. rhombeus than to Korean A. rhombeus. Divergence of Korean A. rhombeus and A. barbatulus from Japanese A. rhombeus was estimated to be from the late Pliocene (3.44 Mya) and the early Pleistocene (1.98 Mya), respectively. Each event closely coincided with the time of the Japan Sea opening. Japanese A. rhombeus comprised seven lineages: three in Honshu and four in Kyushu. One lineage in central Kyushu was genetically closer to the Honshu lineages than to other lineages in northern Kyushu. Divergence of Japanese lineages was estimated to be from the early to middle Pleistocene (0.55-0.93 Mya), during a period of geological and paleoclimatic change, including volcanic activity. Population expansion in the late Pleistocene (<0.10 Ma) was suggested in many of the lineages, which accords with other freshwater fishes. Biogeographically the ancestral A. rhombeus/A. barbatulus was likely to have repeatedly colonized Japan from the continent through land bridges in the late Pliocene and the early Pleistocene. However, the close genetic relationship between Japanese A. rhombeus and A. barbatulus suggests another possibility, with the second colonization occurring in reverse, from Japan to China. The small genetic distance between them indicates that the colonization occurred later than colonization events of other freshwater fishes, including other bitterling species.

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion.

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

Involvement of a Membrane-Bound Amphiphilic Helix in Substrate Discrimination and Binding by an Escherichia coli S2P Peptidase RseP.

Intramembrane proteases (IMPs) are a unique class of proteases that catalyze the proteolysis within the membrane and regulate diverse cellular processes in various organisms. RseP, an Escherichia coli site-2 protease (S2P) family IMP, is involved in the regulation of an extracytoplasmic stress response through the cleavage of membrane-spanning anti-stress-response transcription factor (anti-σE) protein RseA. Extracytoplasmic stresses trigger a sequential cleavage of RseA, in which first DegS cleaves off its periplasmic domain, and RseP catalyzes the second cleavage of RseA. The two tandem-arranged periplasmic PDZ (PDZ tandem) domains of RseP serve as a size-exclusion filter which prevents the access of an intact RseA into the active site of RseP IMP domain. However, RseP's substrate recognition mechanism is not fully understood. Here, we found that a periplasmic region of RseP, located downstream of the PDZ tandem, contains a segment (named H1) predicted to form an amphiphilic helix. Bacterial S2P homologs with various numbers of PDZ domains have a similar amphiphilic helix in the corresponding region. We demonstrated that the H1 segment forms a partially membrane-embedded amphiphilic helix on the periplasmic surface of the membrane. Systematic and random mutagenesis analyses revealed that the H1 helix is important for the stability and proteolytic function of RseP and that mutations in the H1 segment can affect the PDZ-mediated substrate discrimination. Cross-linking experiments suggested that H1 directly interacts with the DegS-cleaved form of RseA. We propose that H1 acts as an adaptor required for proper arrangement of the PDZ tandem domain to perform its filter function and for substrate positioning for its efficient cleavage.

28S rDNA haplotypes of males are distinct from those of androgenetic hermaphrodites in the clam Corbicula leana.

The clam Corbicula leana exists in two forms, hermaphrodites and males. Our previous study on mitochondrial DNA suggested that the male nuclear DNA might have derived from hermaphrodite C. leana relatively recently. To clarify the origin of males in the clam, sequences of the nuclear 28S rDNA divergent domain (which is 441-444 bp long) in androgenetic hermaphrodites and males and dioecious (bisexual) species were analyzed. Unexpectedly, the nuclear 28S rDNA haplotypes of males and hermaphrodites were distinct. Haplotype network analysis indicated that males and hermaphrodites are reproductively isolated from each other without sharing the same nuclear haplotype. These results support a hypothesis that the egg nuclear genome of androgenetic hermaphrodites is replaced by the male sperm genome, and only males develop after fertilization by a male spermatozoon.