The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation, whereas FGF-10 promotes receptor recycling and cell migration. By combining mass-spectrometry-based quantitative proteomics with fluorescence microscopy and biochemical methods, we find that FGF-10 specifically induces the rapid phosphorylation of tyrosine (Y) 734 on FGFR2b, which leads to PI3K and SH3BP4 recruitment. This complex is crucial for FGFR2b recycling and responses, given that FGF-10 stimulation of either FGFR2b_Y734F mutant- or SH3BP4-depleted cells switches the receptor endocytic route to degradation, resulting in decreased breast cancer cell migration and the inhibition of epithelial branching in mouse lung explants. Altogether, these results identify an intriguing ligand-dependent mechanism for the control of receptor fate and cellular outputs that may explain the pathogenic role of deregulated FGFR2b, thus offering therapeutic opportunities.

RNF111/Arkadia targets SUMOylated XPC for ubiquitylation, negatively regulating its association with damaged DNA

Many bactericide species express surface proteins that interact with human serum albumin (HSA). Protein PAB from the anaerobic bacterium Finegoldia magna (formerly Peptostreptococcus magnus) represents one of these proteins. Protein PAB contains a domain of 53 amino acid residues known as the GA module. GA homologs are also found in protein G of group C and G streptococci. Here we report the crystal structure of HSA in complex with the GA module of protein PAB. The model of the complex was refined to a resolution of 2.7 Å and reveals a novel binding epitope located in domain II of the albumin molecule. The GA module is composed of a left-handed threehelix bundle, and residues from the second helix and the loops surrounding it were found to be involved in HSA binding. Furthermore, the presence of HSAbound fatty acids seems to influence HSA-GA complex formation. F. magna has a much more restricted host specificity compared with C and G streptococci, which is also reflected in the binding of different animal albumins by proteins PAB and G. The structure of the HSA-GA complex offers a molecular explanation to this unusually clear example of bacterial adaptation.

M proteins and other members of the M protein family, expressed on the surface of Streptococcus pyogenes, bind host proteins such as immunoglobulins, albumin, and fibrinogen. Protein H and the M1 protein are expressed by adjacent genes and both belong to the M protein family. In this work, the structure and stability of these two proteins have been investigated. As judged from sequence analysis and circular dichroism spectroscopy, the proteins are almost entirely in an alpha-helix conformation. The amino acids are arranged in a seven-residue (heptad) repeat pattern along the greater part of the proteins. These observations support the previously accepted model of M proteins as coiled-coil dimers. However, it was also found that the structures of both proteins were thermally unstable; i.e., the content of helix conformation was greatly reduced at 37 degrees C as compared to 25 degrees C or below. Together with previous findings that these proteins appear as monomers at 37 degrees C and dimers at low temperatures, the results suggest that the coiled-coil dimers are unfolded at 37 degrees C. The heptad patterns of protein H and the M1 protein showed a nonoptimal distribution of residues expected for a coiled-coil conformation. This is a possible explanation for the low thermal stability of the proteins. It was also demonstrated that the proteins were stabilized in the presence of the ligands IgG and/or albumin. Protein H and M1 protein show a high degree of sequence similarity in their C-terminal regions, and a fragment from this region displayed a high content of helix conformation, whereas fragments from the nonsimilar N-terminal parts did not adopt any stable folded structure. Thus, the C-terminal parts, which are conserved within the M protein family, may constitute a framework for the formation of the parallel helical coiled-coil structure, and we propose that the less stable N-terminal part may also participate in antiparallel interaction with M proteins on adjacent bacteria. The results suggest that temperature fluctuations in the environment could change the properties of bacterial surface proteins, thereby affecting the molecular interactions between the bacterium and its host.

SUMOylation of the ubiquitin ligase HERC2 promotes efficient chromatin licensing in the vicinity of DNA double-strand breaks.

Protein L is a multidomain protein expressed at the surface of some strains of the anaerobic bacterial species Peptostreptococcus magnus. It has affinity for immunoglobulin (Ig) through interaction with framework structures in the variable Ig light chain domain. The Ig-binding activity is located to five homologous repeats called B1-B5 in the N-terminal part of the protein. We have determined the three-dimensional solution structure of the 76 amino acid residue long B1 domain using NMR spectroscopy and distance geometry-restrained simulated annealing. The domain is composed of a 15 amino acid residue long disordered N-terminus followed by a folded portion comprising an alpha-helix packed against a four-stranded beta-sheet. These secondary structural elements are well determined with a backbone atomic root mean square deviation from their mean of 0.54 A. The B domains of protein L show very limited sequence homology to the domains of streptococcal protein G interacting with the heavy chains of IgG. However, despite this fact, and their different binding properties, the fold of the B1 domain was found to be similar to the fold of the IgG-binding protein G domains [Wikström, M., Sjöbring, U., Kastern, W., Björck, L., Drakenberg, T., & Forsén, S. (1993) Biochemistry 32, 3381-3386]. In the present study, the solution structure of the B1 domain enabled a more detailed comparison which can explain the different Ig-binding specificities of these two bacterial surface proteins. Among the differences observed, the alpha-helix orientation is the most striking.(ABSTRACT TRUNCATED AT 250 WORDS)

We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.In the complex molecular interplay between a pathogen and its human host, protein-protein interactions play important roles. For instance, bacteria express surface proteins that interact with abundant human extracellular proteins with high affinity and specificity. In human plasma, albumin (HSA) 1 and immunoglobulins (Ig) are the quantitatively dominating proteins, and significant human pathogens have developed surface proteins that bind these and other plasma proteins (for references, see Ref 1). Two of the most well known such proteins are protein A of Staphylococcus aureus (2) and protein G of group C and G streptococci (3, 4), which both bind to the Fc region of IgG. In 1980, Myhre and Kronvall (5) reported that HSA could bind to the surface of various streptococcal species, including group C and G streptococci. It was later found that protein G was responsible also for the interaction with HSA (6). Protein G has separate binding domains for IgG and HSA (7,8), and as a result C and G streptococci are in vivo covered with an inner layer of IgG and an outer layer of HSA. Peptostreptococcus magnus are strictly anaerobic bacteria that are part of the indigenous human flora of the skin, oral cavity, and gastrointestinal and urogenital tracts. Some isolates of this species bind HSA (9), and notably these isolates are mostly from patients with deep wound infections (10), suggesting that HSA binding turns the commensal P. magnus into a potential pathogen. The surface protein of P. magnus binding HSA is called PAB, and it contains a domain of 45 residues showing a high degree of sequence homology with the HSA binding domains of protein G (11). Analysis of the gene encoding PAB suggested that this domain originates from protein G and has been transferred to and introduced into the pab gene through the action of a conjugational plasmid (related to pCF10 of Enterococcus faecalis) followed by a recombinational event (11). This interspecies exchange of a structurally well defined motif repre...

AFX is a human forkhead transcription factor. Based on results from studies of the orthologous transcription factor DAF-16 in Caenorhabditis elegans, it was suggested that some of the metabolic defects in both type I and type II diabetes may be due to unregulated activity of AFX. In the present study, we report the high-resolution NMR solution structure of the DNA binding domain of AFX. It is the first structure of the DNA binding domain from a small subfamily of forkhead transcription factors (i.e., AFX, FKHR, FKHRL1, FKHRL1P1, and FKHRP1). Despite rather low sequence identity for a protein within the forkhead family, the structure is remarkably similar to those of the DNA binding domains of HNF3-gamma and FREAC-11, and to a lesser extent the DNA binding domain of Genesis which displays a slightly altered orientation of the DNA recognition helix. The high degree of structural similarity between the DNA binding domains of different forkhead transcription factors implies that the repositioning of helix 3, observed for Genesis, cannot be a general feature for modulation of the DNA binding specificity. Other mechanisms that could influence the DNA binding specificity are discussed.