RESUMEN
Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le(b)) and Le(y) antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the ß-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.
Asunto(s)
Antígenos de Grupos Sanguíneos/inmunología , Infecciones por Caliciviridae/inmunología , Evolución Molecular , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Norovirus/química , Sitios de Unión , Antígenos de Grupos Sanguíneos/química , Infecciones por Caliciviridae/virología , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Humanos , Antígenos del Grupo Sanguíneo de Lewis/química , Norovirus/inmunología , Norovirus/patogenicidad , Unión Proteica , Proteínas Virales/química , Proteínas Virales/inmunologíaRESUMEN
Mycobacterium tuberculosis (Mtb) uses maltose-1-phosphate to synthesize α-glucans that make up the major component of its outer capsular layer. Maltose kinase (MaK) catalyzes phosphorylation of maltose. The molecular basis for this phosphorylation is currently not understood. Here, we describe the first crystal structure of MtbMaK refined to 2.4 Å resolution. The bi-modular architecture of MtbMaK reveals a remarkably unique N-lobe. An extended sheet protrudes into ligand binding pocket of an adjacent monomer and contributes residues critical for kinase activity. Structure of the complex of MtbMaK bound with maltose reveals that maltose binds in a shallow cavity of the C-lobe. Structural constraints permit phosphorylation of α-maltose only. Surprisingly, instead of a Gly-rich loop, MtbMaK employs 'EQS' loop to tether ATP. Notably, this loop is conserved across all MaK homologues. Structures of MtbMaK presented here unveil features that are markedly different from other kinases and support the scaffolding role proposed for this kinase.
Asunto(s)
Maltosa/metabolismo , Proteínas de Transporte de Monosacáridos/química , Mycobacterium tuberculosis/enzimología , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Fosforilación , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The tripartite motif-containing protein 2 (TRIM2) functions as an E3 ubiquitin ligase. Loss of function of TRIM2 has been shown to result in early-onset axonal neuropathy. As a member of the TRIM-NHL family of proteins, TRIM2 has a conserved modular architecture that includes N-terminal RING finger and B-box domains, a middle coiled-coil domain and a C-terminal NHL domain. To characterize the functional role of its NHL domain from the perspective of structural biology, a truncation of human TRIM2 (residues 465-744) was expressed, purified and crystallized. Rod-shaped crystals were obtained that diffracted X-rays to 1.7 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 43.6, b = 76.4, c = 107.4 Å, α = 90.0, ß = 94.0, γ = 90.0°. A Matthews coefficient of 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.6%, indicated the presence of three molecules per asymmetric unit, which was further confirmed by the phasing solution from molecular replacement.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/aislamiento & purificación , Cristalización , Humanos , Proteínas Nucleares/química , Estructura Terciaria de Proteína/fisiología , Difracción de Rayos XRESUMEN
The guanine-nucleotide exchange factor (GEF) RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP, thus regulating various downstream cellular processes. RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain, followed by a PXXP motif and a C-terminal pleckstrin homology (PH) domain. The Cdc25 domain of RalGPS1a, which shares about 30% sequence identity with other Cdc25-domain proteins, is thought to be directly engaged in binding and activating the substrate Ral protein. Here we report the crystal structure of the Cdc25 domain of RalGPS1a. The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1. The most remarkable difference between these three Cdc25 domains lies in their active sites, referred to as the helical hairpin region. Consistent with previous enzymological studies, the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding. A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex. However, analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.