title: High affinity, paralog-specific recognition of the Mena EVH1 domain by a miniature protein.
abstract: Many protein domains involved in cell signaling contain or interact with proline-rich sequences, and the design of molecules that perturb signaling pathways represents a foremost goal of chemical biology. Previously we described a protein design strategy in which the well-folded alpha-helix in avian pancreatic polypeptide (aPP) presents short alpha-helical recognition epitopes. The miniature proteins designed in this way recognize even shallow protein clefts with high affinity and specificity. Here we show that the well-folded type-II polyproline helix in aPP can present the short PPII-helical recognition epitope within the ActA protein of Listeria monocytogenes. Like miniature proteins that use an alpha-helix for protein recognition, the miniature protein designed in this way displays high affinity for a natural ActA target, the EVH1 domain Mena1-112, and achieves the elusive goal of paralog specificity, discriminating well between EVH1 domains Mena1-112, VASP1-115, and Evl1-112. Most importantly, the miniature protein competed with ActA in Xenopus laevis egg cytoplasmic extracts, decreasing actin-dependent motility of L. monocytogenes and causing extreme speed variations and discontinuous tail formation. Our results suggest that miniature proteins based on aPP may represent an excellent framework for the design of ligands that differentiate the roles of EVH1 domains in vitro and in vivo.
authors.affiliation: Departments of Chemistry and Molecular, Cellular, Yale University, New Haven, Connecticut 06511, USA.
authors.forename: Dasantila
authors.initials: D
authors.lastname: Golemi-Kotra
authors.forename: Rachel
authors.initials: R
authors.lastname: Mahaffy
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Footer
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: Holtzman
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pollard
authors.forename: Julie A
authors.initials: JA
authors.lastname: Theriot
authors.forename: Alanna
authors.initials: A
authors.lastname: Schepartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology; D003598:Cytoskeletal Proteins; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D008089:Listeria monocytogenes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000276:immunology; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D014982:Xenopus laevis
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C102176:Enah protein, mouse; D000939:Epitopes; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; C072980:actA protein, Listeria monocytogenes; D011392:Proline