Bacteria exploit surface proteins to adhere to other bacteria, surfaces and host cells. Such proteins need to project away from the bacterial surface and resist significant mechanical forces. SasG is a protein that forms extended fibrils on the surface of Staphylococcus aureus and promotes host adherence and biofilm formation. Here we show that although monomeric and lacking covalent cross-links, SasG maintains a highly extended conformation in solution. This extension is mediated through obligate folding cooperativity of the intrinsically disordered E domains that couple non-adjacent G5 domains thermodynamically, forming interfaces that are more stable than the domains themselves. Thus, counterintuitively, the elongation of the protein appears to be dependent on the inherent instability of its domains. The remarkable mechanical strength of SasG arises from tandemly arrayed ‘clamp' motifs within the folded domains. Our findings reveal an elegant minimal solution for the assembly of monomeric mechano-resistant tethers of variable length.

The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNA Phe and tRNATrp show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160°rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids ("binding signatures") together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal "recognition" domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.dihydrouridine synthase | tRNA modification | protein-RNA interaction | substrate specificity | X-ray crystallography D uring the posttranscriptional maturation of tRNA, about 10% of its nucleosides are enzymatically modified at specific positions (1). Altered levels of tRNA modification have been linked to several disorders including cancers (2-8). One of the most common modified nucleosides, dihydrouridine, is produced by reduction of the C5-C6 double bond in uridine. The resulting nonplanar base cannot form stabilizing stacking interactions with neighboring nucleotides and favors the C2′-endo ribose conformation (9). In Escherichia coli, dihydrouridine is commonly found at positions 16, 17, 20, and 20a of the D loop of tRNA (Fig. S1A). The formation of dihydrouridine is catalyzed by dihydrouridine synthases (Dus) (10-12). Different Dus subfamilies display specificity toward distinct subsets of target uridines in tRNA. Whereas the specificity of the four Saccharomyces cerevisiae Dus enzymes has been established (13), little is known about the three E. coli Dus proteins (DusA, DusB, and DusC), except that the specificities are nonoverlapping and that DusA modifies U20 (10). The mechanistic basis of the exquisite substrate specificity of Dus is an intriguing problem because the target uridines are exposed at spatially distinct faces of folded tRNAs, and yet all Dus subfamilies are predicted to adopt the same fold with highly conserved active-site residues (14-16).A stru...

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5Ј-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain. Keywords:Biotin protein ligase; biotin holoenzyme synthetase; ATP binding; protein-protein interactions; posttranslational modification Biotin protein ligase (EC 6.3.4.15), also known as holocarboxylase synthetase, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier domain of biotin-dependent carboxylases in a two-step reaction:This reaction is conserved throughout biology, with the enzymes from different species able to recognize and correctly biotinylate carboxylases from widely divergent sources (Cronan and Wallace 1995;Leon-Del-Rio et al. 1995;Tissot et al. 1996), albeit with differing affinities (Polyak et al. 2001). The biotin carboxylases catalyze key reactions in essential metabolic processes and, thus, not only these enzymes but also the biotin ligases, are essential for survival (Chapman-Smith and Cronan 1999a

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein’s size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-017-1689-9) contains supplementary material, which is available to authorized users.

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