Staphylococcus aureus Sortase A transpeptidase. Calcium promotes sorting signal binding by altering the mobility and structure of an active site loop.
Many virulence factors in gram-positive bacteria are covalently anchored to the cell-wall peptidoglycan by sortase enzymes, a group of widely distributed cysteine transpeptidases. The Staphylococcus aureus Sortase A protein (SrtA) is the archetypal member of the Sortase family and is activated by Ca2+, an adaptation that may facilitate host colonization as elevated concentrations of this ion are encountered in human tissue. Here we show that a single Ca2+ ion bound to an ordered pocket on SrtA allosterically activates catalysis by modulating both the structure and dynamics of a large active site loop. Detailed nitrogen-15 relaxation measurements indicate that Ca2+ may facilitate the adaptive recognition of the substrate by inducing slow micro- to millisecond time-scale dynamics in the active site. Interestingly, relaxation compensated Carr-Purcell-Meiboom-Gill experiments suggest that the time scale of these motions is directly correlated with ion binding. The results of site-directed mutagenesis indicate that this motional coupling is mediated by the side chain of Glu-171, which is positioned within the beta6/beta7 loop and shown to contribute to Ca2+ binding. The available structural and dynamics data are compatible with a loop closure model of Ca2+ activation, in which the beta6/beta7 loop fluctuates between a binding competent closed form that is stabilized by Ca2+, and an open, highly flexible state that removes key substrate contacting residues from the active site.
Naik
Mandar T
MT
Department of Chemistry & Biochemistry, University of California, Los Angeles, California 90095-1570, USA.
Suree
Nuttee
N
Ilangovan
Udayar
U
Liew
Chu Kong
CK
Thieu
William
W
Campbell
Dean O
DO
Clemens
Jeremy J
JJ
Jung
Michael E
ME
Clubb
Robert T
RT
D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000502:physiology*; D003546:Cysteine Endopeptidases; D017931:DNA Primers; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010458:Peptidyl Transferases / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013211:Staphylococcus aureus / Q000201:enzymology*; D014774:Virulence
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D010458:Peptidyl Transferases; D003546:Cysteine Endopeptidases; D002118:Calcium