Biochemistry

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains 4-5 FnI and 3 FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes 3 FnIII and disrupts the observed 4-5 FnI-3 FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-...

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, 6 FnI 1-2 FnII 7-9 FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains 8-9 FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains 6 FnI 1-2 FnII 7 FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains 2 FnII and 7 FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the 8-9 FnI⅐collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.

Integrins play a fundamental role in cell migration and adhesion; knowledge of how they are regulated and controlled is vital for understanding these processes. Recent work showed that Dok1 negatively regulates integrin activation, presumably by competition with talin. To ...

Circadian clocks are widespread endogenous mechanisms that control the temporal pattern of diverse biological processes, including gene transcription. KaiA is the positive element of the cyanobacterial clock because KaiA overexpression elevates ...

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

The extracellular matrix (ECM) is a highly organized multimolecular structure, essential for life in higher organisms. Although substantial high-resolution structural information is available for relatively small fragments of ECM components, the inherent difficulty in preparing and analyzing samples of large, fibrous polymers impedes structural efforts. Here, we review recent advances in understanding the structure of three important ECM components: collagen, fibrillin and fibronectin. Emphasis is placed on the key role of intermolecular interactions in assembling larger, mm scale, structures.

Understanding the mechanisms that coordinate the orientation of cell division planes during embryogenesis and morphogenesis is a fundamental problem in developmental biology. Here we show that the orphan receptor lat-1, a homolog of vertebrate latrophilins, plays an essential role in the establishment of tissue polarity in the C. elegans embryo. We provide evidence that lat-1 is required for the alignment of cell division planes to the anterior-posterior axis and acts in parallel to known polarity and morphogenesis signals. lat-1 is a member of the Adhesion-GPCR protein family and is structurally related to flamingo/CELSR, an essential component of the planar cell polarity pathway. We dissect the molecular requirements of lat-1 signaling and implicate lat-1 in an anteriorposterior tissue polarity pathway in the pre-morphogenesis stage of C. elegans development.

Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as 3 FnI, 5 FnI, 7 FnI and 9 FnI, respectively. We have previously reported that mutation of IGD motifs in modules 7 FnI and 9 FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in 3 FnI and 5 FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within 1-5 FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in 7 FnI and 9 FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, 6 FnI 1-2 FnII 7-9 FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains 8-9 FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains 6 FnI 1-2 FnII 7 FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains 2 FnII and 7 FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the 8-9 FnI⅐collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.

Background: The fibronectin (FN)-collagen interaction is important for cell adhesion and migration. Results: FN modules 8-9 FnI interact with two distinct sites in both chains of collagen I. All six collagen-binding FN modules interact cooperatively with a single collagen site. Conclusion: Collagen I possesses four equipotent sites for FN. Significance: We have mapped FN binding to collagen I and demonstrated the first cooperative interaction. Despite its biological importance, the interaction between fibronectin (FN) and collagen, two abundant and crucial tissue components, has not been well characterized on a structural level. Here, we analyzed the four interactions formed between epitopes of collagen type I and the collagen-binding fragment (gelatin-binding domain (GBD)) of human FN using solution NMR, fluorescence, and small angle x-ray scattering methods. Collagen association with FN modules 8-9 FnI occurs through a conserved structural mechanism but exhibits a 400-fold disparity in affinity between collagen sites. This disparity is reduced in the full-length GBD, as 6 FnI 1-2 FnII 7 FnI binds a specific collagen epitope next to the weakest 8-9 FnI-binding site. The cooperative engagement of all GBD modules with collagen results in four broadly equipotent FN-collagen interaction sites. Collagen association stabilizes a distinct monomeric GBD conformation in solution, giving further evidence to the view that FN fragments form well defined functional and structural units.

Integrins play a fundamental role in cell migration and adhesion; knowledge of how they are regulated and controlled is vital for understanding these processes. Recent work showed that Dok1 negatively regulates integrin activation, presumably by competition with talin. To understand how this occurs, we used NMR spectroscopy and x-ray crystallography to investigate the molecular details of interactions with integrins. The binding affinities of ␤3 integrin tails for the Dok1 and talin phosphotyrosine binding domains were quantified using 15 N-1 H heteronuclear single quantum correlation titrations, revealing that the unphosphorylated integrin tail binds more strongly to talin than Dok1. Chemical shift mapping showed that unlike talin, Dok1 exclusively interacts with the canonical NPXY motif of the ␤3 integrin tail. Upon phosphorylation of Tyr 747 in the ␤3 integrin tail, however, Dok1 then binds much more strongly than talin. Thus, we show that phosphorylation of Tyr 747 provides a switch for integrin ligand binding. This switch may represent an in vivo mechanism for control of integrin receptor activation. These results have implications for the control of integrin signaling by proteins containing phosphotyrosine binding domains.

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the 8 F1 9 F1 module pair. We also show that UR inhibits binding of a peptide from the ␣1 chain of type I collagen to 8 F1 9 F1 and that UR binding to 8 F1 is likely to occur through anti-parallel ␤-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD-and GBD-binding sites form a highly unusual extended tandem ␤-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR-and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn. * This research was supported by the BBSRC.

The chemical shifts of 13 C2 of adenosine residues of DNA were observed to experience a through-space or transhydrogen bond isotope effect as a result of deuterium substitution at the imino hydrogen site of base-paired thymidine residues. NMR measurements of several self-complementary DNA duplexes at natural abundance 13 C in 50% H 2 O, 50% D 2 O solvent mixtures yielded an average trans-hydrogen bond isotope effect, 2h 13 C2, of −47 ppb. The data suggest that stronger hydrogen bonds have more negative 2h 13 C2 values, which means that A:T N1• • •H3 hydrogen bonds increase the anharmonicity of the effective vibrational potential of H3. However, 2h 13 C2 values do not correlate with intra-residue 2 13 C4 values of thymidine observed here and earlier (Vakonakis et al., 2003), which suggests that 2h 13 C2 is not determined entirely by hydrogen bond strength. Instead, the variations observed in 2h 13 C2 values suggest that they may also be sensitive to base pair geometry.

Ribosome synthesis in eukaryotes requires a multitude of transacting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm. In contrast to the well-studied transacting factors, little is known about the contribution of the ribosomal proteins to ribosome biogenesis. Herein, we have analysed the role of ribosomal protein Rpl3p in 60S ribosomal subunit biogenesis. In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability of early and intermediate pre-ribosomal particles, as evidenced by the low steady-state levels of 27SA 3 , 27SB S and 7S L/S precursors. Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles. Interestingly, flow cytometry analysis indicates that Rpl3p-depleted cells arrest in the G1 phase. Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented.

KaiA is a two-domain circadian clock protein in cyanobacteria, acting as the positive element in a feedback loop that sustains the oscillation. The structure of the N-terminal domain of KaiA is that of a pseudo-receiver, similar to those of bacterial response regulators, which likely interacts with components of the clock-resetting pathway. The C-terminal domain of KaiA is highly conserved among cyanobacteria and enhances the autokinase activity of KaiC. Here we present the NMR structure of the C-terminal domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. This domain adopts a novel all ␣-helical homodimeric structure. Several mutations known to affect the period of the circadian oscillator are shown to be located at an exposed groove near the dimer interface. This NMR structure and a 21-Å-resolution electron microscopy structure of the hexameric KaiC particle allow us to postulate a mode of KaiA-KaiC interaction, in which KaiA binds a linker region connecting two globular KaiC domains.

Circadian clocks are widespread endogenous mechanisms that control the temporal pattern of diverse biological processes, including gene transcription. KaiA is the positive element of the cyanobacterial clock because KaiA overexpression elevates transcription levels of clock components. Recently, we showed that the structure of KaiA is that of a domain-swapped homodimer. The N-terminal domain is a pseudo-receiver; thus, it is likely to be involved in signal transduction in the clock-resetting pathway. The C-terminal domain of KaiA is structurally novel and enhances the KaiC autokinase activity directly. Here, we report the NMR structure of the C-terminal domain of KaiA (ThKaiA180C) in complex with a KaiC-derived peptide from the cyanobacterium Thermosynechococcus elongatus BP-1. The protein-peptide interface is revealed to be different from a model that was proposed earlier, is stabilized by a combination of hydrophobic and electrostatic interactions, and includes many residues known to produce a circadian-period phenotype upon substitution. Although the structure of the monomeric subunit of ThKaiA180C is largely unchanged upon peptide binding, the intersubunit dimerization angle changes. It is proposed that modulation of the C-terminal KaiA domain dimerization angle regulates KaiA-KaiC interactions. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1SUY and 1SV1). Backbone and side-chain chemical shifts of ThKaiA180C and CIIABD have been deposited in the BioMagResBank (accession no. 6174).