RESUMEN
Signal transducing adapter molecule (STAM) forms the endosomal sorting complex required for transport-0 (ESCRT-0) complex with hepatocyte growth factor-regulated substrate (Hrs) to sort the ubiquitinated cargo proteins from the early endosomes to the ESCRT-1 complex. ESCRT-0 complex, STAM and Hrs, contains multiple ubiquitin binding domains, in which STAM has two ubiquitin binding domains, Vps27/Hrs/Stam (VHS) and ubiquitin interacting motif (UIM) at its N-terminus. By the cooperation of the multiple ubiquitin binding domains, the ESCRT-0 complex recognizes poly-ubiquitin, especially Lys63-linked ubiquitin. Here, we report the backbone resonance assignments and the secondary structure of the N-terminal 191 amino acids of the human STAM1 which includes the VHS domain and UIM. The {(1)H}-(15)N heteronuclear NOE experiments revealed that an unstructured and flexible loop region connects the VHS domain and UIM. Our work provides the basic information for the further NMR investigation of the interaction between STAM1 and poly-ubiquitin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Isótopos de Carbono , Humanos , Hidrógeno , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Interaction between the signal-transducing adapter molecule 1 (STAM1) Vps27/Hrs/Stam (VHS) domain and ubiquitin was investigated by nuclear magnetic resonance (NMR) spectroscopy. NMR evidence showed that the structure of STAM1 VHS domain resembles that of other VHS domains, especially the homologous domain of STAM2. We found that the VHS domain binds to ubiquitin via its hydrophobic patch consisting of N-terminus of helix 2 and C-terminus of helix 4 in which Trp26 on helix 2 plays a pivotal role in the binding. The binding between VHS and ubiquitin seems to be very similar to that between ubiquitin associated domain (UBA) and ubiquitin, however, the direction of alpha-helices involved in the ubiquitin binding is opposite. Here, we propose a novel ubiquitin binding site and the manner of ubiquitin recognition of the STAM1 VHS domain.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Sitios de Unión , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/genética , Dominios y Motivos de Interacción de Proteínas/genética , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Triptófano/metabolismo , Ubiquitina/química , Ubiquitina/genéticaRESUMEN
The conformational preferences and the solution structure of AnxII(N31), a peptide corresponding to the full-length sequence (residues 1-31) of the human annexin II N-terminal tail domain, were investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. CD results showed that AnxII(N31) adopts a mainly alpha-helical conformation in hydrophobic or membrane-mimetic environments, while a predominantly random structure is adopted in aqueous buffer. In contrast to previous results of the annexin I N-terminal domain peptide [Yoon et al. (2000) FEBS Lett. 484, 241-245], calcium ions showed no effect on the structure of AnxII(N31). The NMR-derived structure of AnxII(N31) in 50% TFE/water mixture showed a horseshoe-like fold comprising the N-terminal amphipathic alpha-helix, the following loop, and the C-terminal helical region. Together, the results establish the first detailed structural data on the N-terminal tail domain of annexin II, and suggest the possibility of the domain to undergo Ca(2+)-independent membrane-binding.