RESUMO
CDC25 homology domain (CDC25-HD) containing Guanine Nucleotide Exchange Factors (GEFs) initiate signalling by small G-proteins of the Ras-family. Each GEF acts on a small subset of the G-proteins only, thus providing signalling selectivity. Rlf is a GEF with selectivity for the G-proteins RalA and RalB. Here the crystal structure of Rlf in complex with Ral is determined. The Rlf·Ral complex crystallised into two different crystal forms, which represent different steps of the exchange reaction. Thereby general insight in the CDC25-HD catalysed nucleotide exchange is obtained. In addition, the basis for the selectivity of the interaction is investigated. The exchange activity is monitored by the use of recombinant proteins. Selectivity determinants in the binding interface are identified and confirmed by a mutational study.
Assuntos
Proteínas de Drosophila/química , Fatores de Transcrição/química , Proteínas ral de Ligação ao GTP/química , Cristalografia por Raios X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismoRESUMO
Rlf is a guanine nucleotide exchange factor for the small G-proteins RalA and RalB and couples Ras- to Ral-signalling. Here the crystal structure of the catalytic module of Rlf consisting of a REM- and a CDC25-homology domain is determined. The structure is distinguished by an extended three stranded ß-sheet called the flagpole. The flagpole is a conserved element in the RalGDS family of guanine nucleotide exchange factors and stabilises the orientation of the REM-domain relative to the CDC25-homology domain. A proline-rich sequence in the flagpole is unique to Rlf and several proteins that interact with this sequence by SH3 domains are identified. Conformational pre-selection results in a gain of affinity and contributes to the establishment of SH3 domain selectivity.
Assuntos
Fatores de Transcrição/química , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina , Humanos , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Miosina Tipo I/química , Fragmentos de Peptídeos/química , Fosfolipase C gama/química , Ligação Proteica , Estrutura Secundária de Proteína , Domínios de Homologia de srcRESUMO
The Ras family of small G-proteins plays an essential role in the regulation of a variety of signal transduction processes, ranging from cell cycle control to the regulation of exocytosis. Signalling by the Ras G-proteins is initiated by the CDC25 homology domain (CDC25-HD) containing guanine nucleotide exchange factors (GEFs); each GEF, with its specific selectivity profile towards G-proteins, commonly acts on only a small subset of the Ras family members. Thus, GEFs play a pivotal part in establishing the activation of the downstream signalling routes. The structural basis for the establishment of selectivity in the GEF-G-protein interaction is only partially understood, and several controversies on the selectivity of GEFs are discussed in the literature. In the present study, we undertook a systematic approach to determine the selectivity of CDC25-HD for members of the Ras family. We generated a data set of 126 pairs using a standardised in vitro approach encompassing purified recombinant proteins, and a comprehensive mutational study analysed the basis of the selectivity. Together, these data highlight the distinct selectivity of various GEFs and allow for predictions of untested combinations of GEFs and G-proteins.
