RESUMO
Plants utilize multiple isoforms of villin, an F-actin regulating protein with an N-terminal gelsolin-like core and a distinct C-terminal headpiece domain. Unlike their vertebrate homologues, plant villins have a much longer linker polypeptide connecting the core and headpiece. Moreover, the linker-headpiece connection region in plant villins lacks sequence homology to the vertebrate villin sequences. It is unknown to what extent the plant villin headpiece structure and function resemble those of the well-studied vertebrate counterparts. Here we present the first solution NMR structure and backbone dynamics characterization of a headpiece from plants, villin isoform 4 from Arabidopsis thaliana. The villin 4 headpiece is a 63-residue domain (V4HP63) that adopts a typical headpiece fold with an aromatics core and a tryptophan-centered hydrophobic cap within its C-terminal subdomain. However, V4HP63 has a distinct N-terminal subdomain fold as well as a novel, high mobility loop due to the insertion of serine residue in the canonical sequence that follows the variable length loop in headpiece sequences. The domain binds actin filaments with micromolar affinity, like the vertebrate analogues. However, the V4HP63 surface charge pattern is novel and lacks certain features previously thought necessary for high-affinity F-actin binding. Utilizing the updated criteria for strong F-actin binding, we predict that the headpiece domains of all other villin isoforms in A. thaliana have high affinity for F-actin.
Assuntos
Actinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Proteínas dos Microfilamentos/metabolismo , Isoformas de Proteínas/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Biopolímeros/química , Biopolímeros/metabolismo , Cromatografia em Gel , Proteínas dos Microfilamentos/química , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Isoformas de Proteínas/química , Propriedades de SuperfícieRESUMO
Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortaseâ A homologues. From these studies, we identified sortaseâ A enzymes that recognize multiple substrates that are unreactive toward sortaseâ A from S.â aureus. We further exploited the ability of sortaseâ A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site-specific modification of the N-terminal serine residue in the naturally occurring antimicrobial peptide DCD-1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S.â pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.