3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy

Abstract

Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three-dimensional (3D) structure of an unstable, low-abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortase A (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site-specific cysteine-reactive paramagnetic tag that does not react with the active-site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non-equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.

A Reactive, Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

“…This is significantly faster than the previously reported 4PhSO2‐PyMTA, DO3MA‐Py, and DO8M‐Py tags . The increased reactivity of DO3MA‐3BrPy‐Gd III towards solvent exposed cysteines, imparted by the Br substitution is consistent with earlier findings …”

Resonance Assignments of Lowly Populated and Unstable Enzyme Intermediate Complex under Real‐Time Conditions

“…The complex decays gradually in solution (Figure ). We have previously assigned many cross‐peaks corresponding to the thioester intermediate complex by comparison of the chemical shift of thioester analogue, which formed through a disulfide bond between C184 of SrtA and QALPECG . However, several residues close to the active site and substrate‐binding pocket in SrtA could not be assigned due to structural variations between the thioester complex and its disulfide analogue (Figure ).…”

“…In the mixture of SrtA and calcium, small new cross‐peaks were determined immediately after the addition of peptide substrate, QALPETG‐NH 2 , and the concentration of the new species was about 20 % that of the major species in solution (Figures , , and S3–S6). To resolve the new cross‐peaks, 15 N‐Thr‐labeled SrtA D82C sample was site‐specifically labeled with a rigid 4PS‐3BrPyMTA tag (Figure S2) that was able to track the thioester complex by means of NMR spectroscopy …”

“…Following a similar procedure, a number of selectively 15 N‐amino acid‐labeled SrtA, including 15 N‐Ile, 15 N‐Leu, 15 N‐Val, and 15 N‐Cys, were reacted with substrate QALPETG‐NH 2 and 15 N HSQC spectra were recorded accordingly (Figures and S3–S6). By comparison with the uniformly 15 N‐labeled protein and its BrPyMTA tag conjugates, most cross‐peaks with large CSPs that could not be assigned by using the thioester analogue were well resolved by using the method proposed herein (Figure S7). Notably, some residues show significant differences in PCSs between the thioester complex and free protein (Figure S7); this suggests the presence of structural variations between the thioester complex and free protein.…”

The MPS1 kinase NTE region has helical propensity and preferred conformations towards the TPR domain

“…Additional modeling is required to better characterize the conformational space. Various approaches have been developed over the past decades [10][11][12][13]. Maximum Occurrence analysis, for instance, provides the maximum percent of time that a conformer can exist by assigning a value for each conformer [9].…”