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Wrch1/RhoU is an atypical member of the Rho family. A major structural difference is the extended N-terminus of Wrch1 (nWrch1) containing three putative SH3 domain-binding motifs whose specificities are unknown. To define the impact of this extended region on coupling Wrch1 to cellular signaling, we analyzed in this study nWrch1 interaction with Src homology 3 (SH3) domains of different adaptor proteins. Using sedimentation and isothermal titration calorimetric (ITC) measurements, we identified isolated SH3 domains of growth factor receptor-bound protein 2 (Grb2), noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), c-Src, chicken tumor virus no. 10 (CT 10) regulator kinase 1 (Crk1), and p120 as low-affinity Wrch1-binding partners. Interestingly, under cell-based conditions, nWrch1 bound tightly to endogenous Grb2 and Nck, but not to Crk, c-Src, or p120. Consistent with this, a very tight nWrch1 interaction with full-length Grb2 and Nck1 was confirmed in vitro by ITC measurements indicating that high avidity of the adaptor proteins can compensate for the low affinity of their SH3 domains. Peptide analysis revealed that the central PxxP motif of nWrch1, which employs a minimal consensus sequence of eight amino acids with an essential arginine next to the PxxP motif, is responsible for these interactions. Thus, novel functional insights from this study suggest that multiple upstream signals may converge on Wrch1 directly through its SH3 domain-binding properties. [ABSTRACT FROM AUTHOR]

Abstract: The mechanism through which apidaecin (GNNRPVYIPQPRPPHPRL) like proline rich, non-helical, antibacterial peptides penetrate into the bacterial cells is not yet clearly understood. To comprehend their transport across the bacterial cells, a detailed structure–activity correlation of apidaecin and its selected analogs is undertaken. In membrane like environment apidaecin exhibits a structural change which is mislaid in its biologically inactive P11 →Q substitution analog. This new structure, acquired by apidaecin but not by P11 →Q might be responsible for the difference in their antibacterial response. With this suggestion we explored the membrane permeation response of both by incubating them with small unilamellar vesicles (SUV). Unlike apidaecin, the P11 →Q did not induce leakage from SUV. To confirm whether this response is due to the substitution of P11 →Q of PQP motif, we chose P-ab (YVPLPNVPQPGRRPFPT), an N-terminal domain of abaecin which is homologous to apidaecin in terms of all prolines including conserved PQP, for comparison. Unlike P11 →Q but like apidaecin, P-ab also permeablized the SUV. Computational analysis also indicated that this particular mutation has a strong structural impact. These results led us to hypothesize that in bacterial environment apidaecin undergoes an ordered structural change that facilitates its entry into the bacterial membrane and also that PXP motives are important for this structural change. Apidaecin analogs not viable to organize/transform into this functionally active conformation are deleteriously affected. Adaptation to a unique conformation though insufficient (since functional binding with intracellular targets is also mandatory) seems to be an important prerequisite for the manifestation of full spectrum of antibacterial activity of apidaecin like peptides. [Copyright &y& Elsevier]