Figure 4.
The active site of SrtAΔN59 and its calcium-binding site. (A) Expanded view of the active site. As for the papain/cathepsin family, N98, H120, and C184 may form a catalytic triad that mediates the transpeptidation reaction and are positioned within a large hydrophobic pocket suitable for sorting signal binding. (B) Representative histogram of the Cα-carbon chemical shift differences between the calcium-free and -bound forms of sortase plotted as a function of residue number. These data and observed chemical shift changes in the amide nitrogen and proton atoms were used to determine the calcium-binding surface of sortase. The largest chemical shift changes occur in amino acids V168, L169, and N114; they are 2.1 (Cα), 7.3 (N), and 1.3 (HN) ppm, respectively. We estimate that calcium binds to sortase with a Kd of ≈10−4 to 10−3 M. The secondary structure of sortase is indicated. The absence of a bar indicates that the chemical shift of the α-carbon is unassigned in either the calcium-bound or calcium-free form. (C) Ribbon drawing of sortase showing the putative calcium-binding site and calcium-sensitive active-site loop. Three acidic side chains (E105, E108, D112) are poised to bind calcium as judged by localized, large-amplitude calcium-dependent changes in their chemical shifts and in surrounding amino acids. Several amino acids distal to the calcium-binding site exhibit broadening of their backbone atoms only in the presence of calcium (indicated by magenta spheres). These amino acids experience micro- to millisecond fluctuations in their magnetic environments, which presumably result either from calcium-dependent movements of the loop connecting strands β6 and β7 or from the binding of a second cation. The active-site side chains of H120, C184, and W194 are shown for reference. (D) Electrostatic surface of the SrtAΔN59 active site shown in a similar orientation as in C (acidic and basic surfaces are colored red and blue, respectively.) An active-site groove can readily accommodate a polypeptide substrate denoted by a white line. The hypothetical positioning of the sorting signal is derived by analogy with the papain/cathepsin protease family.