A very small number of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophysical characterization and X-ray crystal structure determination show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approximately 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic experiments suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy-connecting two monomers of an intertwined homodimer into a single protein chainsupports a model for evolution of knotted structures via gene duplication.Anfinsen | energy landscape | folding kinetics | protein folding | topology

Gram-positive bacteria use sortase cysteine transpeptidase enzymes to covalently attach proteins to their cell wall and to assemble pili. In pathogenic bacteria sortases are potential drug targets, as many of the proteins that they display on the microbial surface play key roles in the infection process. Moreover, the Staphylococcus aureus Sortase A (SaSrtA) enzyme has been developed into a valuable biochemical reagent because of its ability to ligate biomolecules together in vitro via a covalent peptide bond. Here we review what is known about the structures and catalytic mechanism of sortase enzymes. Based on their primary sequences, most sortase homologs can be classified into six distinct subfamilies, called class A–F enzymes. Atomic structures reveal unique, class-specific variations that support alternate substrate specificities, while structures of sortase enzymes bound to sorting signal mimics shed light onto the molecular basis of substrate recognition. The results of computational studies are reviewed that provide insight into how key reaction intermediates are stabilized during catalysis, as well as the mechanism and dynamics of substrate recognition. Lastly, the reported in vitro activities of sortases are compared, revealing that the transpeptidation activity of SaSrtA is at least 20-fold faster than other sortases that have thus far been characterized. Together, the results of the structural, computational, and biochemical studies discussed in this review begin to reveal how sortases decorate the microbial surface with proteins and pili, and may facilitate ongoing efforts to discover therapeutically useful small molecule inhibitors.

Background: Sortase enzymes catalyze a transpeptidation reaction that displays bacterial surface proteins. Results: Structural and computational studies reveal how the sortase B enzyme recognizes its sorting signal substrate. Conclusion: Sortase enzymes catalyze transpeptidation using a substrate-stabilized oxyanion hole. Significance: The results of this work could facilitate the rational design of sortase inhibitors.

Bacillus anthracis forms metabolically dormant endospores that upon germination can cause lethal anthrax disease in humans. Efficient sporulation requires the activity of the SrtC sortase (BaSrtC), a cysteine transpeptidase that covalently attaches the BasH and BasI proteins to the peptidoglycan of the forespore and predivisional cell, respectively. To gain insight into the molecular basis of protein display, we used nuclear magnetic resonance to determine the structure and backbone dynamics of the catalytic domain of BaSrtC (residues Ser56–Lys198). The backbone and heavy atom coordinates of structurally ordered amino acids have coordinate precision of 0.42 ± 0.07 and 0.82 ± 0.05 Å, respectively. BaSrtCΔ55 adopts an eight-stranded β-barrel fold that contains two short helices positioned on opposite sides of the protein. Surprisingly, the protein dimerizes and contains an extensive, structurally disordered surface that is positioned adjacent to the active site. The surface is formed by two loops (β2–β3 and β4–H1 loops) that surround the active site histidine, suggesting that they may play a key role in associating BaSrtC with its lipid II substrate. BaSrtC anchors proteins bearing a noncanonical LPNTA sorting signal. Modeling studies suggest that the enzyme recognizes this substrate using a rigid binding pocket and reveals the presence of a conserved subsite for the signal. This first structure of a class D member of the sortase superfamily unveils class-specific features that may facilitate ongoing efforts to discover sortase inhibitors for the treatment of bacterial infections.

Many species of Gram-positive bacteria use sortase enzymes to assemble long, proteinaceous pili structures that project from the cell surface to mediate microbial adhesion. Sortases construct highly stable structures by catalyzing a transpeptidation reaction that covalently links pilin subunits together via isopeptide bonds. Most Gram-positive pili are assembled by class C sortases that contain a “lid”, a structurally unique N-terminal extension that occludes the active site. It has been hypothesized that the “lid” in many sortases is mobile, and thus capable of readily being displaced from the enzyme to facilitate substrate binding. Here, we show using NMR dynamics measurements, in vitro assays, and molecular dynamics simulations that the lid in the class C sortase from Streptococcus pneumoniae (SrtC1) adopts a rigid conformation in solution that is devoid of large magnitude conformational excursions that occur on mechanistically relevant timescales. Additionally, we show that point mutations in the lid induce dynamic behavior that correlates with increased hydrolytic activity and sorting signal substrate access to the active site cysteine residue. These results suggest that the lid of the S. pneumoniae SrtC1 enzyme has a negative regulatory function, and imply that a significant energetic barrier must be surmounted by as of yet unidentified factors to dislodge it from the active site to initiate pilus biogenesis.