RESUMO
The search for general strategies for inhibiting protein-protein interactions has been stimulated by recognition of the key role they play in virtually every process of living systems. Multiprotein complex assembly and localization by PDZ domain-containing proteins exemplify processes critical to cell physiology and function that are mediated by beta strand association. Here we describe the development of substituted "@-tides," protease-resistant peptidomimetics incorporating conformationally restricted amino acid surrogates that reproduce the hydrogen-bonding pattern and side-chain functionality of a beta strand. The synthetic flexibility and generality of the substituted @-tide design was demonstrated by the synthesis of a panel of ligands for the alpha1-syntrophin PDZ domain. The rational design of a small molecule of unprecedented affinity for the PDZ domain suggests that these peptidomimetics may provide a general method for inhibiting protein-protein interactions involving extended peptide chains.
Assuntos
Ligantes , Biomimética , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Estrutura Molecular , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína/fisiologiaRESUMO
Familial hypercholanemia (FHC) is characterized by elevated serum bile acid concentrations, itching, and fat malabsorption. We show here that FHC in Amish individuals is associated with mutations in tight junction protein 2 (encoded by TJP2, also known as ZO-2) and bile acid Coenzyme A: amino acid N-acyltransferase (encoded by BAAT). The mutation of TJP2, which occurs in the first PDZ domain, reduces domain stability and ligand binding in vitro. We noted a morphological change in hepatic tight junctions. The mutation of BAAT, a bile acid-conjugating enzyme, abrogates enzyme activity; serum of individuals homozygous with respect to this mutation contains only unconjugated bile acids. Mutations in both TJP2 and BAAT may disrupt bile acid transport and circulation. Inheritance seems to be oligogenic, with genotype at BAAT modifying penetrance in individuals homozygous with respect to the mutation in TJP2.
Assuntos
Aciltransferases/genética , Ácidos e Sais Biliares/sangue , Síndromes de Malabsorção/sangue , Síndromes de Malabsorção/genética , Proteínas de Membrana/genética , Mutação , Estudos de Casos e Controles , Etnicidade/genética , Feminino , Genótipo , Humanos , Desequilíbrio de Ligação , Fígado/patologia , Síndromes de Malabsorção/patologia , Masculino , Linhagem , Pennsylvania , Fenótipo , Junções Íntimas/patologia , Proteína da Zônula de Oclusão-2RESUMO
PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse alpha1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements.
Assuntos
Proteínas de Membrana/química , Proteínas Musculares/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio , Sequência Conservada/genética , Ligação de Hidrogênio , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Concentração Osmolar , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
LIN-2, -7 (L27) homology domains are putative protein-protein interaction modules found in several scaffold proteins involved in the assembly of polarized cell-signaling structures. These specific interaction pairs are well conserved across metazoan species, from worms to man. We have expressed and purified L27 domains from multiple species and find that certain domains from proteins such as Caenorhabditis elegans LIN-2 and LIN-7 can specifically heterodimerize. Biophysical analysis of interacting L27 domains demonstrates that the domains interact with a 1:1 stoichiometry. Circular dichroism studies reveal that the domains appear to function as an obligate heterodimer; individually the domains are largely unfolded, but when associated they show a significant increase in helicity, as well as a cooperative unfolding transition. These novel obligate interacting pairs are likely to play a key role in regulating the organization of signaling proteins at polarized cell structures.
