The human RPS4 paralogue on Yq11.223 encodes a structurally conserved ribosomal protein and is preferentially expressed during spermatogenesis.

BACKGROUND: The Y chromosome of mammals is particularly prone to accumulate genes related to male fertility. However, the high rate of molecular evolution on this chromosome predicts reduced power to the across-species comparative approach in identifying male-specific genes that are essential for sperm production in humans. We performed a comprehensive analysis of expression of Y-linked transcripts and their X homologues in several human tissues, and in biopsies of infertile patients, in an attempt to identify new testis-specific genes involved in human spermatogenesis. RESULTS: We present evidence that one of the primate-specific Y-linked ribosomal protein genes, RPS4Y2, has restricted expression in testis and prostate, in contrast with its X-linked homologue, which is ubiquitously expressed. Moreover, we have determined by highly specific quantitative real time PCR that RPS4Y2 is more highly expressed in testis biopsies containing germ cells. The in silico analysis of the promoter region of RPS4Y2 revealed several differences relative to RPS4Y1, the more widely expressed paralogue from which Y2 has originated through duplication. Finally, through comparative modelling we obtained the three dimensional models of the human S4 proteins, revealing a conserved structure. Interestingly, RPS4Y2 shows different inter-domain contacts and the potential to establish specific interactions. CONCLUSIONS: These results suggest that one of the Y-linked copies of the ribosomal protein S4 is preferentially expressed during spermatogenesis and might be important for germ cell development. Even though RPS4Y2 has accumulated several amino acid changes following its duplication from RPS4Y1, approximately 35 million years ago, the evolution of the Y-encoded RPS4 proteins is structurally constrained. However, the exclusive expression pattern of RPS4Y2 and the novelties acquired at the C-terminus of the protein may indicate some degree of functional specialisation of this protein in spermatogenesis.

Autonomous folding of interdomain regions of a modular polyketide synthase.

Domains within the multienzyme polyketide synthases are linked by noncatalytic sequences of variable length and unknown function. Recently, the crystal structure was reported of a portion of the linker between the acyltransferase (AT) and ketoreductase (KR) domains from module 1 of the erythromycin synthase (6-deoxyerythronolide B synthase), as a pseudodimer with the adjacent ketoreductase (KR). On the basis of this structure, the homologous linker region between the dehydratase (DH) and enoyl reductase (ER) domains in fully reducing modules has been proposed to occupy a position on the periphery of the polyketide synthases complex, as in porcine fatty acid synthase. We report here the expression and characterization of the same region of the 6-deoxyerythronolide B synthase module 1 AT-KR linker, without the adjacent KR domain (termed DeltaN AT1-KR1), as well as the corresponding section of the DH-ER linker. The linkers fold autonomously and are well structured. However, analytical gel filtration and ultracentrifugation analysis independently show that DeltaN AT1-KR1 is homodimeric in solution; site-directed mutagenesis further demonstrates that linker self-association is compatible with the formation of a linker-KR pseudodimer. Our data also strongly indicate that the DH-ER linker associates with the upstream DH domain. Both of these findings are incompatible with the proposed model for polyketide synthase architecture, suggesting that it is premature to allocate the linker regions to a position in the multienzymes based on the solved structure of animal fatty acid synthase.

Structural basis of hepatocyte growth factor/scatter factor and MET signalling.

The polypeptide growth factor, hepatocyte growth factor/scatter factor (HGF/SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF/SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryo-electron microscopy, we show that conversion of pro(single-chain)HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF/SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF/SF contact the face of the seven-blade beta-propeller domain of MET harboring the loops connecting the beta-strands b-c and d-a, whereas the C-terminal serine proteinase homology domain binds the opposite "b" face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF/SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF/SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF/SF-MET signaling.

Amino acid sequence, biochemical characterization, and comparative modeling of a nonspecific lipid transfer protein from Amaranthus hypochondriacus.

Plant nonspecific lipid transfer proteins (nsLTPs) are characterized by their ability to bind a broad range of hydrophobic ligands in vitro. Their biological function has not yet been elucidated, but they could play a major role in plant defense to physical and biological stress. An nsLTP was isolated from Amaranthus hypochondriacus seeds and purified by gel filtration and reversed-phase high-performance liquid chromatography techniques. The molecular mass of the protein as determined by mass spectrometry is 9747.29 Da. Data from amino acid sequence, circular dichroism and binding/displacement of a fluorescent lipid revealed that it belongs to the nsLTP1 family. The protein shows the alpha-helical secondary structure typical for plant nsLTPs 1 and shares 40 to 57% sequence identity with nsLTPs 1 from other plant species and 100% identity with an nsLTP1 from Amaranthus caudatus. A model structure of the protein in complex with stearate based on known structures of maize and rice nsLTPs 1 suggests a protein fold complexed with lipids closely related to that of maize nsLTP1.

Functional map and domain structure of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor.

Little is known about the large ectodomain of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor/scatter factor (HGF/SF). Here, we establish by deletion mutagenesis that the HGF/SF and heparin-binding sites of MET are contained within a large N-terminal domain spanning the alpha-chain (amino acids 25-307) and the first 212 amino acids of the beta-chain (amino acids 308-519). Neither the cystine-rich domain (amino acids 520-561) nor the C-terminal half of MET (amino acids 562-932) bind HGF/SF or heparin directly. The MET ectodomain, which behaves as a rod-shaped monomer with a large Stokes radius in solution, binds HGF/SF in the absence or presence of heparin, and forms a stable HGF/SF-heparin-MET complex with 1:1:1 stoichiometry. We also show that the ligand-binding domain adopts a beta-propeller fold, which is similar to the N-terminal domain of alphaV integrin, and that the C-terminal half contains four Ig domains (amino acids 563-654, 657-738, 742-836, and 839-924) of the unusual structural E set, which could be modeled on bacterial enzymes. Our studies provide 3D models and a functional map of the MET ectodomain. They have broad implications for structure-function of the MET receptor and the related semaphorin and plexin proteins.