Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition.

Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end.

Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632.

Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability.

Molecular mechanism for the regulation of rho-kinase by dimerization and its inhibition by fasudil.

Rho-kinase is a key regulator of cytoskeletal events and a promising drug target in the treatment of vascular diseases and neurological disorders. Unlike other protein kinases, Rho-kinase requires both N- and C-terminal extension segments outside the kinase domain for activity, although the details of this requirement have been elusive. The crystal structure of an active Rho-kinase fragment containing the kinase domain and both the extensions revealed a head-to-head homodimer through the N-terminal extension forming a helix bundle that structurally integrates the C-terminal extension. This structural organization enables binding of the C-terminal hydrophobic motif to the N-terminal lobe, which defines the correct disposition of helix alphaC that is important for the catalytic activity. The bound inhibitor fasudil significantly alters the conformation and, consequently, the mode of interaction with the catalytic cleft that contains local structural changes. Thus, both kinase and drug conformational pliability and stability confer selectivity.

Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells.

The low density lipoprotein receptor-related protein 4 gene (Lrp4) was identified by subtractive screening of cDNAs of the migratory primordial germ cells (PGCs) of E8.5-9.5 embryo and E3.5 blastocysts. Lrp4 is expressed in PGCs in the hindgut and the dorsal mesentery of E9.5 embryos, and in germ cells in the genital ridges of male and female E10.5-13.5 embryos. Lrp4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of Lrp4 expression in the blastocyst, embryonic stem cells and embryonic germ cells suggests the Lrp4 is a molecular marker that distinguishes the germ cells from embryo-derived pluripotent stem cells.

Regulation of expression of mouse interferon-induced transmembrane protein like gene-3, Ifitm3 (mil-1, fragilis), in germ cells.

Mouse interferon-induced transmembrane protein (IFITM) gene, Ifitm3 (previously known as mil-1 and fragilis), is expressed in primordial germ cells (PGCs), in their precursors, and in germ cells of the fetal gonads (Saitou et al. [2002] Nature 418:293-300; Tanaka and Matsui [2002] Mech Dev 119S:S261-S267). By examining the expression of green fluorescent protein transgene under the control of DNA sequences flanking exon 1, we have identified domains that direct Ifitm3 transcription in PGCs and their precursors in gastrula stage and 13.5 days post coitum embryos. Germ cell-specific expression is achieved by the activity of a consensus element unique to the Ifitm genes, which may act to suppress Ifitm3 expression in somatic tissues. The lack of any influence of the interferon-stimulable response elements on transgene expression in the germ-line suggests that interferon-mediated response is not critical for activating Ifitm3.