β5/β6 hairpin chemical shift differences and J_eff(0) values for the two-states of CBAP-acylated BlaR^S.

<p>β5/β6 hairpin chemical shift differences and J_eff(0) values for the two-states of CBAP-acylated BlaR^S.</p

Model of the CBAP ring flip in CBAP-acylated BlaR^S.

<p>(A) The degrees of rotational freedom of the CBAP biphenyl substituent. Green sticks correspond to the bound conformation of CBAP in the x-ray crystal structure, PDB 3Q7Z. Yellow and blue sticks correspond to racemization of the biphenyl and rotation of the entire biphenyl unit, respectively. (B) Proposed rotation of the CBAP biphenyl demonstrates a steric clash–indicated with an asterisk–with the β5/β6 hairpin that would necessitate a conformational change.</p

Slow exchange and amide CSPs from CBAP-acylation of BlaR^S.

<p>(A) Residues demonstrating slow chemical exchange (orange spheres) and significant CSPs (blue spheres) are mapped onto PDB 3Q7Z. Spheres indicate residues assigned in both apo and CBAP-acylated BlaR^S. Green sticks represent the bound β-lactam CBAP. (B) Pronounced ¹⁵N^H-¹H exchange squares of V532 and Y536 of U-[¹⁵N] 80% deuterated CBAP-acylated BlaR^S. Spectra were recorded at 18.8 T and T(nom) = 293.8 K; ¹⁵N TROSY (Blue) versus EXSY-R₁ TROSY (black) using a 400 ms exchange relaxation delay.</p

Structure of the CBAP-acylated BlaR1 sensor domain.

<p>Ribbon representation of BlaR^S acylated by CBAP (PDB code 3Q7Z). Three conserved structural features include the β5/β6 hairpin (red), the P-loop (slate), the Ω-loop (magenta). Conserved sequence motifs include the “S-x-x-K” (Orange, S389-T390-Y391-K392), the “S-x-N/D” (Cyan, S437-V438-N439), and the “K-T/S-G” (yellow, K526-T527-G528) motifs. The bound β-lactam CBAP is indicated by Green sticks. The β7-Helix K turn is indicated in black.</p

Chemical structure of CBAP.

<p>Chemical structure of CBAP.</p

Changes in J_eff(0) due to acylation of BlaR^S by CBAP.

<p>Comparison of μs-ms or ps-ns dynamics between apo and CBAP-acylated BlaR^S using reduced spectral density mapping. The parameter J_eff(0) was compared using a dimensionless ΔJ(0) ratio. Spheres indicate residues whose assignments are both known and can be compared between apo and CBAP-acylated BlaR^S. Residues whose dimensionless ΔJ(0) ratios greater than two standard deviations of the core average are indicated by red (positive) and blue (negative) spheres. Positive ratios correspond to enhanced μs-ms or reduced ps-ns dynamics; negative ratios correspond to reduced μs-ms or enhanced ps-ns dynamics.</p

Interdomain Interactions Support Interdomain Communication in Human Pin1

Pin1 is an essential mitotic regulator consisting of a peptidyl–prolyl isomerase (PPIase) domain flexibly tethered to a smaller Trp–Trp (WW) binding domain. Communication between these domains is important for Pin1 in vivo activity; however, the atomic basis for this communication has remained elusive. Our previous nuclear magnetic resonance (NMR) studies of Pin1 functional dynamics suggested that weak interdomain contacts within Pin1 enable allosteric communication between the domain interface and the distal active site of the PPIase domain., A necessary condition for this hypothesis is that the intrinsic properties of the PPIase domain should be sensitive to interdomain contact. Here, we test this sensitivity by generating a Pin1 mutant, I28A, which weakens the wild-type interdomain contact while maintaining the overall folds of the two domains. Using NMR, we show that I28A leads to altered substrate binding affinity and isomerase activity. Moreover, I28A causes long-range perturbations to conformational flexibility in both domains, for both the apo and substrate-complexed states of the protein. These results show that the distribution of conformations sampled by the PPIase domain is sensitive to interdomain contact and strengthen the hypothesis that such contact supports interdomain allosteric communication in Pin1. Other modular systems may exploit interdomain interactions in a similar manner

Revealing Cell-Surface Intramolecular Interactions in the BlaR1 Protein of Methicillin-Resistant <i>Staphylococcus aureus</i> by NMR Spectroscopy

In methicillin-resistant <i>Staphylococcus aureus</i>, β-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

Investigation of Signal Transduction Routes within the Sensor/Transducer Protein BlaR1 of <i>Staphylococcus aureus</i>

The transmembrane antibiotic sensor/signal transducer protein BlaR1 is part of a cohort of proteins that confer β-lactam antibiotic resistance in methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) [Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) <i>Chem. Rev. 105</i>, 395–424; Llarrull, L. I., Fisher, J. F., and Mobashery, S. (2009) <i>Antimicrob. Agents Chemother. 53</i>, 4051–4063; Llarrull, L. I., Toth, M., Champion, M. M., and Mobashery, S. (2011) <i>J. Biol. Chem. 286</i>, 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 (BlaR^S). The acylation activates the cytoplasmic protease domain through an obscure signal transduction mechanism. Here, we compare protein dynamics of apo versus antibiotic-acylated BlaR^S 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