RESUMEN
We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.
Asunto(s)
Adenosina Trifosfato/metabolismo , Difosfatos/metabolismo , Péptido Sintasas/metabolismo , Adenosina Trifosfato/química , Sitios de Unión , Biotecnología/métodos , Dominio Catalítico , Técnicas Químicas Combinatorias/métodos , Difosfatos/química , Cinética , Modelos Químicos , Estructura Molecular , Reproducibilidad de los Resultados , Especificidad por Sustrato , Tirocidina/química , Tirocidina/metabolismoRESUMEN
The dramatic improvement in the NMR spectra of insulin-like growth factor I (IGF-I) in the presence of a peptide identified from a phage display library has allowed for the first time the determination of a high-resolution solution structure for much of IGF-I. The three helices of IGF-I in this complex have an arrangement similar to that seen in high-resolution crystal structures of IGF-I and insulin, although there are differences in the conformation and precise location of helix 3. A cluster of hydrophobic and basic side chains within the turn-helix motif of the peptide contact a hydrophobic patch on helices 1 and 3 of IGF-I. The importance of this patch for tight binding was verified using alanine scanning mutagenesis of the peptide in two different phage display formats. Consistent with its antagonistic activity, the peptide binds to a region implicated by mutagenesis studies to be important for association with IGF binding proteins (IGFBPs). The ability of the peptide to also inhibit signaling has important implications for the manner in which IGF-I interacts with its receptor. Interestingly, the peptide uses the same binding site as detergent and a fragment of IGFBP-5 identified in other IGF-I complexes. The ligand-induced structural variability of helix 3 in these complexes suggests that exchange between such conformations may be the source of the dynamic nature of free IGF-I and likely has functional significance for the ability of IGF-I to recognize two signaling receptors and six binding proteins with high affinity.
Asunto(s)
Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Bacteriófago M13/metabolismo , Unión Competitiva/genética , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Insulina/química , Cinética , Ligandos , Imitación Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Unión Proteica/genética , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
A panel of 22 naïve peptide libraries was constructed in a polyvalent phage display format and sorted against insulin-like growth factor-1 (IGF-1). The libraries were pooled to achieve a total diversity of 4.4 x 10(11). After three rounds of selection, the majority of the phage clones bound specifically to IGF-1, with a disulfide-constrained CX(9)C scaffold dominating the selection. Four monovalently displayed sub-libraries were designed on the basis of these conserved motifs. Sub-library maturation in a monovalent format yielded an antagonistic peptide that inhibited the interactions between IGF-1 and two cell-surface receptors and those between IGF-1 and two soluble IGF binding proteins with micromolar potency. NMR analysis revealed that the peptide is highly structured in the absence of IGF-1, and peptides that preorganize the binding elements were selected during the sorting.