Allosteric Coupling of CARMIL and V-1 Binding to Capping Protein Revealed by Hydrogen-Deuterium Exchange.

Actin assembly is important for cell motility. The ability of actin subunits to join or leave filaments via the barbed end is critical to actin dynamics. Capping protein (CP) binds to barbed ends to prevent subunit gain and loss and is regulated by proteins that include V-1 and CARMIL. V-1 inhibits CP by sterically blocking one binding site for actin. CARMILs bind at a distal site and decrease the affinity of CP for actin, suggested to be caused by conformational changes. We used hydrogen-deuterium exchange with mass spectrometry (HDX-MS) to probe changes in structural dynamics induced by V-1 and CARMIL binding to CP. V-1 and CARMIL induce changes in both proteins' binding sites on the surface of CP, along with a set of internal residues. Both also affect the conformation of CP's ßß subunit "tentacle," a second distal actin-binding site. Concerted regulation of actin assembly by CP occurs through allosteric couplings between CP modulator and actin binding sites.

The C-terminal domain of full-length E. coli SSB is disordered even when bound to DNA.

The crystal structure of full-length homotetrameric single-stranded DNA (ssDNA)-binding protein from Escherichia coli (SSB) has been determined to 3.3 A resolution and reveals that the entire C-terminal domain is disordered even in the presence of ssDNA. To our knowledge, this is the first experimental evidence that the C-terminal domain of SSB may be inherently disordered. The N-terminal DNA-binding domain of the protein is well ordered and is virtually indistinguishable from the previously determined structure of the chymotryptic fragment of SSB (SSBc) in complex with ssDNA. The absence of observable interactions with the core protein and the crystal packing of SSB together suggest that the disordered C-terminal domains likely extend laterally away from the DNA- binding domains, which may facilitate interactions with components of the replication machinery in vivo. The structure also reveals the conservation of molecular contacts between successive tetramers mediated by the L(45) loops as seen in two other crystal forms of SSBc, suggesting a possible functional relevance of this interaction.