RESUMO
Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH-NPF interaction, the solution structures of two EH-NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I beta-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the beta-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.
Assuntos
Asparagina/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Fenilalanina/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Prolina/metabolismo , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Asparagina/química , Sítios de Ligação , Proteínas de Ligação ao Cálcio/síntese química , Ciclização , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fenilalanina/química , Fosfoproteínas/síntese química , Prolina/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Eps15 homology (EH) domains interact with proteins involved in endocytosis and signal transduction. EH domains bind to Asn-Pro-Phe (NPF) consensus motifs of target proteins. A few EH domains, such as the third EH domain (EH(3)) of human Eps15, prefer to bind Phe-Trp (FW) sequences. The structure of EH(3) has been solved by nuclear magnetic resonance (NMR) spectroscopy and is the first of an FW- and NPF-binding EH domain. Both FW and NPF sequences bind in the same hydrophobic pocket as shown by heteronuclear chemical shift mapping. EH(3) contains the dual EF-hand fold characteristic of the EH domain family, but it binds calcium with high affinity in the first EF-hand rather than the usual coordination in the second EF-hand. Point mutations were designed based on differences in the EH(3) and the second EH domain (EH(2)) of human Eps15 that alter the affinity of the domains for FW or NPF motif peptides. Peptides that mimic binding sites in the potential EH(3) targets Rab, synaptojanin, and the cation-dependent mannose 6-phosphate receptor were used to explore wild-type and mutant affinities. Characterization of the structure and binding properties of an FW- and NPF-binding EH domain and comparison to an NPF-specific EH domain provide important insights into the mechanisms of EH domain ligand recognition.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Motivos EF Hand , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/genética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Termodinâmica , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The transition metal complexes [eta(6)- (2beta-carbomethoxy-3beta-phenyltropane)]tricarbonylchromium (3) and [eta(6)-(2beta-carbomethoxy-3beta-phenyltropane)] [eta(5)-(pentamethylcyclopentadienyl)]ruthenium(II)triflate (4) were synthesized from 2beta-carbomethoxy-3beta-phenyltropane (2, WIN 35,065) to further elucidate the influence of substituents on the 3beta-aryl on the affinity of the ligand for cocaine-binding sites at the dopamine transporter. The compounds were tested for their ability to displace bound [(3)H]WIN 35,428 (5) from rat caudate putamen tissue and for their ability to inhibit [(3)H]dopamine uptake. The binding affinity for 3 was 2-fold greater than those observed for cocaine (1) and 2, while the binding affinity for 4 was found to be 100-fold less than those of 1 and 2. In addition, 3 was equipotent with 1 and 2 in [(3)H]dopamine uptake inhibition studies, while 4 was 10-fold less potent. The potencies of the complexes 3 and 4 correlated well with the structure-activity relationships of other 2beta-carbomethoxy-3beta-aryltropane derivatives. These data further support a pharmacophore model in which the region occupied by the aryl ring is a lipophilic pocket with electropositive character.
