RESUMO
Trimeric autotransporter adhesins (TAAs) represent an important class of pathogenicity factors in proteobacteria. Their defining feature is a conserved membrane anchor, which forms a 12-stranded beta-barrel through the outer membrane. The proteins are translocated through the pore of this barrel and, once export is complete, the pore is occluded by a three-stranded coiled coil with canonical heptad (7/2) sequence periodicity. In many TAAs this coiled coil is extended by a segment of varying length, which has pentadecad (15/4) periodicity. We used X-ray crystallography and biochemical methods to analyze the transition between these two periodicities in the coiled-coil stalk of the Yersinia adhesin YadA. Our results show how the strong right-handed supercoil of the 15/4-periodic part locally undergoes further over-winding to 19/5, before switching at a fairly constant rate over 14 residues to the canonical left-handed supercoil of the 7/2-periodic part. The transition region contains two YxD motifs, which are characteristic for right-handed coiled-coil segments of TAAs. This novel coiled-coil motif forms a defined network of inter- and intrahelical hydrogen bonds, thus serving as a structural determinant. Supercoil fluctuations have hitherto been described in coiled coils whose main sequence periodicity is disrupted locally by discontinuities. Here we present the first detailed analysis of two fundamentally different coiled-coil periodicities being accommodated in the same structure.
Assuntos
Adesinas Bacterianas/química , Conformação Proteica , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Yersinia/química , Yersinia/citologiaRESUMO
Most core residues of coiled coils are hydrophobic. Occasional polar residues are thought to lower stability, but impart structural specificity. The coiled coils of trimeric autotransporter adhesins (TAAs) are conspicuous for their large number of polar residues in position d of the core, which often leads to their prediction as natively unstructured regions. The most frequent residue, asparagine (N@d), can occur in runs of up to 19 consecutive heptads, frequently in the motif [I/V]xxNTxx. In the Salmonella TAA, SadA, the core asparagines form rings of interacting residues with the following threonines, grouped around a central anion. This conformation is observed generally in N@d layers from trimeric coiled coils of known structure. Attempts to impose a different register on the motif show that the asparagines orient themselves specifically into the core, even against conflicting information from flanking domains. When engineered into the GCN4 leucine zipper, N@d layers progressively destabilized the structure, but zippers with 3 N@d layers still folded at high concentration. We propose that N@d layers maintain the coiled coils of TAAs in a soluble, export-competent state during autotransport through the outer membrane. More generally, we think that polar motifs that are both periodic and conserved may often reflect special folding requirements, rather than an unstructured state of the mature proteins.
Assuntos
Adesinas Bacterianas/química , Motivos de Aminoácidos , Íons/química , Estrutura Terciária de Proteína , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Asparagina/química , Asparagina/genética , Asparagina/metabolismo , Sítios de Ligação , Transporte Biológico , Dicroísmo Circular , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Íons/metabolismo , Zíper de Leucina , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Multimerização Proteica , Salmonella enterica/genética , Salmonella enterica/metabolismo , Homologia de Sequência de Aminoácidos , Treonina/química , Treonina/genética , Treonina/metabolismoRESUMO
The novel 3H-spiro[1-benzofuran-2-cyclopentan]-3-one skeleton has been made accessible by different routes. One- and two-step protocols lead to tricyclic and tetracyclic benzofuranones 2 and 3, respectively. A four-step synthesis to spirocompound 4 is described. The novel spirocyclic benzofuranones display modest to no inhibition of the human peptidyl prolyl cis/trans isomerase Pin1.
Assuntos
Benzofuranos/síntese química , Benzofuranos/farmacologia , Peptidilprolil Isomerase/antagonistas & inibidores , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Benzofuranos/química , Humanos , Lactonas/química , Peptidilprolil Isomerase de Interação com NIMA , Compostos de Espiro/químicaRESUMO
The peptidyl prolyl cis/trans isomerase Pin1 has been implicated in the development of cancer, Alzheimer's disease and asthma, but highly specific and potent Pin1 inhibitors remain to be identified. Here, by screening a combinatorial peptide library, we identified a series of nanomolar peptidic inhibitors. Nonproteinogenic amino acids, incorporated into 5-mer to 8-mer oligopeptides containing a d-phosphothreonine as a central template, yielded selective inhibitors that blocked cell cycle progression in HeLa cells in a dose-dependent manner.
