The Rab11-Family Interacting Protein (Rab11-FIP) group of effector proteins contain a highly conserved region in their C-termini that bind the GTPase, Rab11. Rab11 belongs to the largest family of small GTPases and is believed to regulate vesicle docking with target membranes and vesicle fusion. The amino acid sequence of the Rab11-FIP proteins predicts coiled-coil formation in the conserved C-terminal domain. In this study on Rab11-FIP2, we found experimental evidence for the coiled-coil and then defined the minimal structured core using limited proteolysis. We also showed that the Rab11-FIP2 coiled-coil domain forms a parallel homodimer in solution using cross-linking and mutagenesis and sedimentation equilibrium experiments. Various constructs representing the Cterminal domain of Rab11-FIP2 were characterized by circular dichroism and their affinity with Rab11 measured using isothermal titration calorimetry. The longest construct was both wellstructured and bound Rab11. A construct truncated at the N-terminus was poorly structured, but retained the same affinity for binding to Rab11. Conformational changes were also demonstrated upon complex formation between Rab11 and Rab11-FIP2. A construct truncated at the C-terminus, which was the minimal coiled-coil domain defined by limited proteolysis, did not retain the ability to interact with Rab11 although it was as well-structured as the longer peptide. These data show that coiled-coil formation and Rab11 binding are separable functions of the C-terminal domain of Rab11-FIP2. The dissection of Rab11 binding from the formation of defined structure in a coiled-coil provides a potential mechanism for regulating Rab11-dependent endosomal trafficking.

Rab11 plays a central role in plasma membrane recycling which returns cellular receptors for re-use at the cell surface. A recently identified family of Rab11 interacting proteins (FIP) includes FIP2. The C-terminal region of FIP2 is essential for colocalization with Rab11 on early endosomes and to enable formation of higher order oligomers. Rab11 binding and oligomerization of FIP2 are separable. Here we have determined the three-dimensional structure of the 40 residue coiled-coil oligomerization domain of FIP2 in the absence of Rab11 using NMR methods. The N-terminal half showed strong NOE cross-peaks and well dispersed NMR resonances, whereas the C-terminal half had fewer NOE cross-peaks and less chemical shift dispersion. The C-terminal 10 residues were mostly disordered. The final structures of the dimer had favorable Ramachandran angles and a rootmean-square deviation of 0.59 ± 0.13 Å over superimposed backbone residues. The structure allows a comparison to a structure of FIP2 in complex with Rab11 that was determined crystallographically. In complex with Rab11, the C-terminal residues are not disordered, but have a helical structure that predicts residual dipolar coupling constants that are incompatible with those measured on the unbound FIP2. In both structures, a histidine residue is found at the normally hydrophobic position of the heptad repeat of the coiled-coil and here we show its ionization destabilizes the coiled-coil structure. Together these data allow us to build a model in which the binding of FIP-family proteins to Rab11 can be described in terms of conformational changes and that suggests new modes of regulation.Eukaryotic cells internalize nutrients, fluids, and other molecules from the extracellular environment through the plasma membrane via a process called endocytosis. The Rab protein family members regulate vesicle tethering and docking with target membranes and ensure the precision of vesicle fusion. In particular, Rab11 plays a central role in plasma membrane recycling which returns cellular receptors for re-use at the surface (1,2).Rab11 conducts its biological effects through interacting with effector proteins in a GTPdependent manner. Rab11 behaves as a typical GTPase protein in which the GTP bound form is active and the GDP bound form is inactive. These two forms have different conformations in their switch 1 and switch 2 regions that are built on a scaffold of a central six-stranded β sheet flanked by two α helices on each side (3). The interacting region of the effector is generally † Supported in part by NIH grant GM067985 ‡ Atomic coordinates have been deposited with the Research Collaboratory for Structural Bioinformatics Protein Databank, filename 2K6S and NMR assignments have been deposited at the BioMagResBank (BMRB) NMR structural database, accession number 15880. a dimer of α-helices arranged in a coiled-coil (4-6) although sometimes a helix is used to form a binding surface, but with structures different from a coiled-coil (7-9).A recently identified family of...

The surface protein SdrE, a microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family protein expressed on the surface of Staphylococcus aureus (S. aureus), can recognize human complement regulator Factor H and C4BP, thus making it a potentially promising vaccine candidate. In this study, SdrE278-591 was found to directly affect S. aureus host cell invasion. Additionally, the crystal structure of SdrE278-591 at a resolution of 1.25 Å was established, with the three-dimensional structure revealing N2-N3 domains which fold in a manner similar to an IgG fold. Furthermore, a putative ligand binding site located at a conserved charged groove formed by the interface between N2 and N3 domains was identified, with β2 suspected to occupy the ligand recognizing site and undergo a structural rearrangement to allow ligand binding. Overall, these findings have further contributed to the understanding of SdrE as a key factor for S. aureus invasivity and will enable a better understanding of bacterial infection processes.