RESUMEN
The transmembrane antibiotic sensor/signal transducer protein BlaR1 is part of a cohort of proteins that confer ß-lactam antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) [Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) Chem. Rev. 105, 395-424; Llarrull, L. I., Fisher, J. F., and Mobashery, S. (2009) Antimicrob. Agents Chemother. 53, 4051-4063; Llarrull, L. I., Toth, M., Champion, M. M., and Mobashery, S. (2011) J. Biol. Chem. 286, 38148-38158]. Specifically, BlaR1 regulates the inducible expression of ß-lactamases that hydrolytically destroy ß-lactam antibiotics. The resistance phenotype starts with ß-lactam antibiotic acylation of the BlaR1 extracellular domain (BlaRS). The acylation activates the cytoplasmic protease domain through an obscure signal transduction mechanism. Here, we compare protein dynamics of apo versus antibiotic-acylated BlaRS using nuclear magnetic resonance. Our analyses reveal inter-residue interactions that relay acylation-induced perturbations within the antibiotic-binding site to the transmembrane helix regions near the membrane surface. These are the first insights into the process of signal transduction by BlaR1.
Asunto(s)
Proteínas Bacterianas/química , Metaloendopeptidasas/química , Staphylococcus aureus Resistente a Meticilina/química , Transducción de Señal , Resistencia betalactámica , Acilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/metabolismo , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de ProteínaRESUMEN
In methicillin-resistant Staphylococcus aureus, ß-lactam antibiotic resistance is mediated by the transmembrane protein BlaR1. The antibiotic sensor domain BlaR(S) and the L2 loop of BlaR1 are on the membrane surface. We used NMR to investigate interactions between BlaR(S) and a water-soluble peptide from L2. This peptide binds BlaR(S) proximal to the antibiotic acylation site as an amphipathic helix. Acylation of BlaR(S) by penicillin G does not disrupt binding. These results suggest a signal transduction mechanism whereby the L2 helix, partially embedded in the membrane, propagates conformational changes caused by BlaR(S) acylation through the membrane via transmembrane segments, leading to antibiotic resistance.
Asunto(s)
Proteínas Bacterianas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Acilación , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína , Transducción de Señal/fisiología , Marcadores de Spin , beta-Lactamas/metabolismoRESUMEN
Drug design involves iterative ligand modifications. For flexible ligands, these modifications often entail restricting conformational flexibility. However, defining optimal restriction strategies can be challenging if the relationship between ligand flexibility and biological activity is unclear. Here, we describe an approach for ligand flexibility-activity studies using Nuclear Magnetic Resonance (NMR) spin relaxation. Specifically, we use (13)C relaxation dispersion measurements to compare site-specific changes in ligand flexibility for a series of related ligands that bind a common macromolecular receptor. The flexibility changes reflect conformational reorganization resulting from formation of the receptor-ligand complex. We demonstrate this approach on three structurally similar but flexibly differentiated ligands of human Pin1, a peptidyl-prolyl isomerase. The approach is able to map the ligand dynamics relevant for activity and expose changes in those dynamics caused by conformational locking. Thus, NMR flexibility-activity studies can provide information to guide strategic ligand rigidification. As such, they help establish an experimental basis for developing flexibility-activity relationships (FAR) to complement traditional structure-activity relationships (SAR) in molecular design.
Asunto(s)
Oligopéptidos/química , Oligopéptidos/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Secuencia de Aminoácidos , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Peptidilprolil Isomerasa de Interacción con NIMA , Relación Estructura-Actividad , TemperaturaRESUMEN
Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain (13)C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene (13)CH(2) and methyl (13)CH(3) groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of mus-ms conformational dynamics related to binding and catalysis. We perform these measurements at natural (13)C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.
Asunto(s)
Isótopos de Carbono/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Secuencias de Aminoácidos , Sitios de Unión , Humanos , Ligandos , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/química , Fosfopéptidos/química , Unión Proteica , Conformación ProteicaRESUMEN
We show that Carr-Purcell-Meiboom-Gill (CPMG) 13Calpha NMR relaxation dispersion measurements are a viable means for profiling mus-ms ligand dynamics involved in receptor binding. Critically, the dispersion is at natural 13C abundance; this matches typical pharmaceutical research settings in which ligand isotope-labeling is often impractical. The dispersion reveals ligand 13Calpha nuclei that experience mus-ms modulation of their chemical shifts due to binding. 13Calpha shifts are dominated by local torsion angles , psi, chi1; hence, these experiments identify flexible torsion angles that may assist complex formation. Since the experiments detect the ligand, they are viable even in the absence of a receptor structure. The mus-ms dynamic information gained helps establish flexibility-activity relationships. We apply these experiments to study the binding of a phospho-peptide substrate ligand to the peptidyl-prolyl isomerase Pin1.