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, 54 (8), 1600-10

Investigation of Signal Transduction Routes Within the Sensor/Transducer Protein BlaR1 of Staphylococcus Aureus

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Investigation of Signal Transduction Routes Within the Sensor/Transducer Protein BlaR1 of Staphylococcus Aureus

Michael W Staude et al. Biochemistry.

Abstract

The transmembrane antibiotic sensor/signal transducer protein BlaR1 is part of a cohort of proteins that confer β-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 β-lactamases that hydrolytically destroy β-lactam antibiotics. The resistance phenotype starts with β-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.

Figures

Figure 1
Figure 1
Overview of BlaR1 activation by β-lactam antibiotics in S aureus. Important steps I–IV are detailed in main text.
Figure 2
Figure 2
NH Chemical-Shift Perturbations of BlaRS induced by penG. Blue shading on the structure are residues with CSP magnitudes > 0.050 (2 standard deviations). Wheat shading represent amides with CSP magnitudes < 0.050. Dark grey shading are residues lacking assignments in one or both states. Orange sticks highlight active-site residues nearest the bound penG. In the bar chart, yellow arrows and red cylinders refer to β-strands (numbers) and α-helices (letters), respectively. The rectangular boxes highlight conserved residue motifs among bacterial acyltransferases (27), S-xx-K (S389, T390, Y391, and K392), S/T-x-N, (S437, V438, and V439), and KT/SG (K526, T527, G528).
Figure 3
Figure 3
Intrinsic backbone flexibility of apo BlaRS, via Jeff(0). Red shading indicates regions where Jeff(0) is higher than average (enhanced exchange dynamics). Marine shading indicates regions where Jeff(0) is lower than the average (enhanced sub-nanosecond bond mobility). Wheat shading indicates regions included in the core-average. Dark grey shading indicates regions lacking data in the apo form.
Figure 4
Figure 4
Changes in NH Jeff(0) caused by penG acylation. (Left panel) Map of the fractional change, ΔJ/JeffAPO(0)=1JeffpenG(0)/JeffAPO(0). Colored shading are sites for which the magnitude of fractional changes was > 0.025 from the core average. Red shading indicates positive values (acylation decreases Jeff(0)) and blue shading indicates negative values (acylation increases Jeff(0). Wheat shading denotes residues with fractional changes consistent with the core average +/− 2 s.d., while dark grey shading indicates residues lacking data in one or both states. (Right panel) Paramagnetic Relaxation Enhancement (PRE) response of amide protons in 15N-labeled BlaRS, upon interaction with spin-labeled L2-short peptide (11). Blue is the strongest interaction site and red is a weaker non-specific interaction.
Figure 5
Figure 5
Amide hydrogen exchange response of BlaRS upon penG acylation. (Left). Map of ΔkH2O/kH2OAPO. Colored shading denotes sites for which the magnitude of the fractional changes > 1 s.d. from the core average. Blue shading indicates residues for which acylation decreases kH2O (negative ΔkH2O/kH2OAPO), and red shading indicates residues for which acylation increases kH2O (positive ΔkH2O/kH2OAPO). Wheat shading indicates residues lacking significant differences in kH2O. Dark grey shading indicates residues lacking data in one or both states. Right). Blue highlights kNOE fractional changes greater than one standard deviation from the core-average (decreased exchange upon acylation). Red highlights residues with kNOE fractional changes less than one standard deviation from the core-average (increased exchange upon acylation). Wheat shading indicates residues lacking significant differences in kNOE. Dark grey shading indicates residues lacking data in one or both states.
Figure 6
Figure 6
Proposed mechanism for BlaR1 signal transduction. Transduction involves a contiguous chain of interactions that include the side-chain substituent of the covalently bound antibiotic, the β5/β6 hairpin, and extracellular loop L2. Our previous results (11) indicated that L2 binds β5/β6 as an amphipathic helix that could partially embed into the membrane. (a) In the apo state, β5/β6 interacts with the L2 helix (b) Acylation of Ser389 by β-lactam antibiotic results in new interactions between the antibiotic side chain and bulky protein side chains of β5/β6, such as Ile531, which then perturb the β5/β6-L2 interaction. The effects propagate from L2 to its connecting transmembrane helix, and onto the cytoplasmic Zn2+-protease domain, stimulating β-lactamase production. (c, d) Full view and zoomed in perspectives of the interaction chain based on PDB 1XA7. Green sticks highlight the bound antibiotic penG (the phenyl moiety of its C6 substituent.) Magenta shading and sticks highlight the β5/β6 region and its residues (I531 and G534). Deep salmon shading denotes the disordered C/D loop that shows local dynamic changes upon acylation. Yellow denotes α-helix K, implicated in cryptic allostery (30). Orange, slate, and aquamarine are S-xx-K, S/T-x-N, and KT/SG motifs conserved among bacterial acyltransferases (27). The proposed interaction between the antibiotic C6 substituent and I531 is supported by the work of Mobashery and co-workers (37), which showed that β-lactam antibiotics with larger substituents are better inducers, and the fact that related proteins that are enzymes (e.g. OXA-10) instead of transducers lack the bulky hydrophobic side chain at this position.

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