No abstract

We report a method based on electrospray ionization (ESI) mass spectrometry to determine solution association constants (K A) for complexes between glycopeptide antibiotics (vancomycin and ristocetin) and several peptide ligands. The measured K A values are in good agreement with previously reported values obtained by standard spectroscopic titration techniques. The pH stability of the ristocetin−diacetyl-l-lysyl-d-alanyl-d-alanine complex was investigated by ESI mass spectrometry and circular dichroism (CD) spectroscopy. The two methods produce very similar results, demonstrating that the ESI mass spectra reflect the pH stability of the complex. In solution, the antibiotics bind stereospecifically to peptides containing a C-terminal d-Ala-d-Ala sequence, whereas no complex formation is observed with peptides containing the l-Ala-l-Ala stereoisomer. To investigate whether electrospray ionization is able to reflect the structurally specific interaction between antibiotics and d-Ala-d-Ala peptide ligands, an equimolar mixture of vancomycin, diacetyl-l-lysyl-d-alanyl-d-alanine, and the deuterium-labeled stereoisomer diacetyl-l-lysyl-l-alanyl-l-alanine-d 6 was analyzed by ESI mass spectrometry. In agreement with solution-phase behavior, only ions corresponding to a complex between diacetyl-l-lysyl-d-alanyl-d-alanine and vancomycin are observed.

Review: 185 refs.

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5b and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.