STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF SORTASE A

Sortases have been known to be essential in Gram-positive bacteria for attaching proteins onto the peptidoglycan layer of the bacterium. Sortase A has been found to be useful as a “molecular stapler”, although; in vivo, the enzyme is responsible for attaching proteins to the peptidoglycan layer of Gram-positive bacteria. It accomplishes both of these tasks by joining two proteins together via an LPXTG sorting sequence. The enzyme has been proven to be very useful in attaching any two proteins together without worrying about recombinant techniques to generate the fusion protein. The problem with this enzyme is that the catalytic diad, which is composed of Cys-184 and His-120, has to be in a certain form that exists .2% of the time at pH 7.0. There is also a hydrolytic shunt that the enzyme can undergo instead of the productive transpeptidase reaction. These issues lead to groups attempting to place S.aureus SrtA through directed evolution in order to increase the catalytic efficiency of the enzyme. Although mutants have been generated that increase the catalytic efficiency 13-fold and 130-fold, the structural basis behind this increase is poorly understood. Using crystallography, we will attempt to discover the structural basis behind the rate enhancement as well as understand more about different species of SrtA. We also will attempt to kinetically characterize the S.aureus SrtA enzyme, its mutants, and different strains of SrtA. Thus far G.moribillorum SrtA has been crystallized and its structure shows that there is a distinction in the β6/β7 loop which has been implied to be important to catalysis. Furthermore, the pentaglycine kinetics shone some light on how the different mutants interact with the pentaglycine substrate of S.aureus SrtA

Rep-DNA complexes and their role in AAV DNA transactions

Adeno-associated Virus (AAV) Rep proteins are multifunctional proteins that carry out various DNA transactions required for the life cycle of AAV. The Rep proteins have been found to be important for genome replication, gene regulation, site-specific integration and play an essential role in genome packaging. There are two main groups of Rep proteins: large and small Reps; both groups are SF3 helicase family members. During DNA packaging, studies have shown that the small Rep proteins are critical to produce fully packed particles. Using stopped-flow kinetic analysis, we show a significant difference in helicase activity between the small and large Rep proteins that support the notion that the small Rep proteins are the primary motor to package DNA due to more efficient motor activity. That leaves the large Rep proteins to serve a different role during packaging. In previous studies, we have shown that the large Rep proteins have the ability to change their oligomeric state depending on the nature of the DNA substrate. We can observe double octameric rings with single-stranded DNA (ssDNA) and heptameric complex with double-stranded DNA (dsDNA). To understand Rep protein structural plasticity, we solved a 6.96 Å cryo-EM structure of Rep68*/ssDNA complex illustrating that the formation of Rep octamer rings is dominated by interactions between their N-terminal origin-binding domain (OBD) using the same interface utilized to recognize dsDNA specifically. Our analysis of the structural data suggests that the double octameric ring structure is stabilized by ssDNA that bridges octameric rings together. The structure shows that the helicase domains are highly flexible and that ssDNA is present at the center of the ring. In addition, we have solved a preliminary 12 Å model of Rep68*/dsDNA complex showing a heptameric ring encircling a DNA molecule. Our structural and functional data offer insights to the various Rep-DNA scaffolds that can perform diverse functions during the AAV life cycle

Directed evolution provides insight into conformational substrate sampling by SrtA

<div><p>The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme’s inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.</p></div

Directed evolution provides insight into conformational substrate sampling by SrtA - Fig 3

<p>A) Schematic of the continuous fluorescence assay used to measure the initial acylation kinetics as previously described. B) Schematic of the fluorescence polarization assay used to follow the complete reaction coordinate. The peptide substrate is labeled at the amino terminus with the fluorescent label tetramethylrhodomain (TMR), which is efficiently excited at 557nm and emits at 576nm. In the absence of SrtA or secondary substrate (biotin labeled GGGGGDYK peptide), the fluorescence polarization is low. Alternatively, following the reaction of substrates in the presence of SrtA, avidin binding greatly increases the increases fluorescence polarization. C) Kinetic parameters for wild-type (WT) or SrtA variants.</p

SrtA is a dynamic protein that undergoes a large conformational change upon substrate binding.

<p>A) Residues selected for mutagenesis for spin-label incorporation are labeled and their positions are shown in the NMR (top) or x-ray crystal structure (bottom) models. B) Distance distribution plots of SrtA variants with pairs of spin labels incorporated at the indicated positions in the absence (solid line) or presence of sort-tag substrate peptide (dashed line). The colored distributions represent the predicted distances obtained from PRONOX using the x-ray (blue; PDB: 1T2W) and NMR models (gray; PDB: 2KID-1). The background-corrected dipolar evolution data (gray dots) are shown for each pair of spin labeled mutant SrtA proteins (100 μM) as recorded on a Q-band Bruker ELEXSYS 580 spectrometer (Fig B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184271#pone.0184271.s001" target="_blank">S1 File</a> for the raw data); the black lines represent the fits to the data in the absence of substrate and the blue lines represent the fits to the data for the SrtA protein in the presence of 10x Abz-CLEPTGG.</p

Schematic representation of the protein complementation assay utilized for directed evolution of SrtA.

<p>A) Murine DHFR is cloned into pET-Duet as two independent fragments consisting of the carboxyl-terminal region fused to three amino-terminal glycines in one open reading frame and amino terminus fused to an LPETG sort-tag in a second open reading frame. Endogenous methionine aminopeptidase (MAP) cleaves the initiating methionine to expose the terminal glycines. B) SrtA enzymatically ligates the two fragments to generate an active DHFR. Endogenous bacterial DHFR is inhibited by the prokaryotic specific trimethoprim. C) Initial culture conditions testing the requirement for SrtA in the DHFR complementation assay. D) Overnight growth of bacteria containing the various assay components in the presence and absence of trimethoprim was monitored using optical density at 600nm, OD₆₀₀. Each trial was completed in duplicate. Cells carrying either the pET vector expressing only the split DHFR, or the pRSF vector driving expression of SrtA fail to grow in the presence of trimethoprim. Alternatively, bacteria expressing a positive control mDHFR (mDHFR-(PC)) with the internal LPETGG sequence, grow robustly in the absence of SrtA, but in the presence of trimethoprim. Similarly, bacteria expressing the split mDHFR gene along with SrtA also grow robustly following an overnight growth in trimethoprim. E) Growth of bacteria on LB-Agar plates containing Ampicillin, Kanamycin, IPTG, and trimethoprim. I) and III) BL21(DE3) cells containing pET-Duet C/N-mDHFR with empty pRSF vector, or II) and IV) BL21(DE3) cells containing pET-Duet C/N-mDHFR with pRSF-SrtA.</p

SrtA conformational changes along the reaction coordinate as depicted in the x-ray crystal structure of SrtA bound to LPETG (shown on the left) or the NMR model bound to LPAT* (shown on the right).

<p>The image in the middle is a model computationally morphed between the two structures. The top panels are in wireframe representation with the key catalytic residues in ball-and-stick format in blue, the three glutamic acids involved in Ca²⁺ chelation in red, and peptide substrate in yellow. The bottom panel is the molecular surface representation of the structure colored according to sequence conservation (among all SrtA family members). Residues that are between 76–100% conserved are shown in red, 51–75% conserved in blue, 26–50% conserved in green, and 0–25% conserved in grey. The structure on the left represents initial substrate binding. Note that the substrate sits in a highly conserved (red) pocket. Following binding, the β6- β7 loops twists and lowers/closes while the β7- β8 loop opens ~10Å in a scissor motion. Collectively, these movements guide the substrate from the top of the enzyme toward the newly formed active-site pocket in the bottom while positioning the glutamic acid residues shown in red for optimum binding of Ca²⁺ and properly orienting the residues in blue for catalysis.</p