40 in Carbapenem-Resistant

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Expanded substrate activity of OXA-24/40 in carbapenem-resistant Acinetobacter baumannii involves enhanced binding loop flexibility Michael Wesbrook Staude, David A. Leonard, and Jeffrey W. Peng Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00806 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

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Expanded substrate activity of OXA-24/40 in carbapenem-resistant Acinetobacter baumannii involves enhanced binding loop flexibility AUTHOR INFORMATION Michael W. Staude1, David A. Leonard2, and Jeffrey W. Peng1* 1

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556

2

Department of Chemistry, Grand Valley State University, Allendale, Michigan 49401

Corresponding Author *Tel: (574) 631-2983; Fax: (574) 631-6652; E-mail: [email protected] Funding Sources No competing financial interests have been declared. This work was supported by NIH grants GM085109 (to JWP), 1R15AI082416 (to DAL) and GAANN Grant P200A130203 (to MWS)

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KEYWORDS. Gram negative, Acinetobacter baumannii, carbapenem, antibiotic resistance, OXA-24/40, NMR, NMR Relaxation, protein dynamics.

ABBREVIATIONS USED 2-D, two-dimensional; MHz, megahertz; HSQC, heteronuclear single quantum correlation; ppm, parts-per-million; NMR, Nuclear Magnetic Resonance; rmsd, root-mean-squared-deviation; CHDL, carbapenem-hydrolyzing Class D β-lactamase; ESBL, Extended Spectrum β-lactamase; NOE, nuclear Overhauser effect; TROSY, transverse relaxation optimized spectroscopy; CSP, chemical shift perturbation.


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ABSTRACT Gram-negative bacteria resist β-lactam antibiotics primarily by deploying βlactamase proteins that hydrolytically destroy the antibiotics. In clinical settings, these bacteria are producing variant β-lactamases with “gain of activity” mutations that inactivate a broader range of β-lactams. Learning how these mutations broaden substrate activity is important for coping with β-lactam resistance. Here, we investigate a gain of activity mutation in OXA-24/40, a carbapenem-hydrolyzing Class D β-lactamase (CHDL) in Acinetobacter baumannii. OXA24/40 was originally active against penicillin and carbapenem classes of β-lactams. But a clinical variant of OXA-24/40, the single-site substitution mutant P227S, has emerged with expanded activity that now includes advanced cephalosporins and the monobactam, aztreonam. Using solution state Nuclear Magnetic Resonance (NMR) spectroscopy, we have compared the sitespecific backbone dynamics of wild-type OXA-24/40 and the P227S variant. P227S changes local backbone flexibility in segments important for both binding and hydrolysis of carbapenem and cephalosporin substrates. Our results suggest that mutation-induced changes in sequencespecific dynamics can expand substrate activity, and thus highlight the role of protein conformational dynamics in antibiotic resistance. To our knowledge, this is the first study of CHDL conformational dynamics by NMR, and its impact in the expansion of β-lactam antibiotic resistance.


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INTRODUCTION Gram-negative pathogenic bacteria are becoming increasingly resistant to a broad range of βlactam antibiotics. A prevalent resistance mechanism involves deployment of β-lactamase proteins that hydrolytically destroy the antibiotics. Of particular concern are the carbapenemhydrolyzing Class D β-lactamases (CHDLs). As their name suggests, these enzymes inactivate carbapenems, a class of β-lactam antibiotics typically administered as drugs of last resort 1, 2. This work focuses on a well-known CHDL, OXA-24/40 3 . The “OXA-#” naming convention applies to all Class D β-lactamases (not just CHDLs), and refers to the strong oxacillinase activity noted in the first studies of Class D β-lactamases 4. OXA-24/40 confers carbapenem resistance in the gram-negative pathogen, Acinetobacter baumannii (A. baumannii) baumannii has become a source of hospital-acquired infections worldwide.

5

.

A.

It targets the

immune-compromised and trauma victims, causing bloodstream infections, urinary tract infections, and ventilator-associated pneumonia 4.

The advent of multi-drug resistant A.

baumannii challenges current antibiotic therapy, and fatalities are not uncommon 6. Originally, OXA-24/40 activity had been restricted to the penicillin and carbapenem classes of β-lactams. However, recent clinical isolates of A. baumannii are now producing “gain-ofactivity” variants of OXA-24/40 capable of destroying a broader spectrum of β-lactams compared to the parent enzyme. These gain-of-function mutations are particularly disturbing given the health risk already posed by A. baumannii. Remarkably, the gain-of-activity can result from merely one or two mutations in a β-hairpin loop near the antibiotic binding site

2, 7, 8

In

particular, P227S in the β5-β6 hairpin loop enables new activity against β-lactam substrates traditionally targeted by the extended spectrum β-lactamases (ESBLs)

9, 10

, including 3rd

generation cephalosporins such as ceftazidime and cefotaxime, and the monobactam aztreonam


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. This new activity raises the disturbing possibility of OXA-24/40 becoming active against all

classes of β-lactams. Curiously, x-ray structures show that both P227 and S227 lack direct contact with the bound carbapenem antibiotic 12. This raises the question of how this single site substitution promotes a gain of activity. Comparisons of OXA-24/40 and P227S x-ray structures also show perturbations in the local β5-β6 conformation

10, 11

. Similar perturbations have been observed in x-ray structures of other

OXA variants binding various β-lactam antibiotics

10-14

. These perturbations suggest the β5-β6

hairpin loops of OXA-24/40 and other CHDLs have an intrinsic flexibility that can influence their substrate specificity. Hence, OXA-24/40 variants with amino acid substitutions in the β5β6, in addition to introducing new side chain groups, could explore a broader range of conformations compatible with a broader range of β-lactam substrates. To investigate this possibility, we report here our comparison of the backbone flexibilities of OXA-24/40 (WT) and its single site substitution mutant, P227S, that has gained extended spectrum β-lactamase (ESBL) activity. To our knowledge, this is the first per-residue study of CHDL conformational flexibility and its potential contribution to expanded β-lactam resistance. Our main findings are as follows: (i) WT OXA-24/40 is flexible on both the picosecond (ps) – nanosecond (ns) time scales, and microsecond (µs) – millisecond (ms) time scales, at conserved regions important for substrate binding and hydrolysis, such as the β5-β6 hairpin loop; (ii) the P227S substitution in the β5-β6 loop changes the local flexibility of β5-β6 and more distal residues important for substrate activity. Our results support a model in which P227S alters the breadth of conformations sampled by β5-β6, thereby providing a basis for its enhanced activity against 3rd generation cephalosporins and the monobactam, aztreonam. Our results raise the


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possibility that changes in sequence-dependent functional dynamics of antibiotic resistance proteins can promote “gain of activity” mutations leading to expanded resistance.

MATERIALS and METHODS Isotopically labeled OXA-24/40. The construct of WT OXA-24/40 in the NdeI and BamHI sites of pET24a has been previously described, 12 and was transformed into BL21 (DE3) E. coli cells. Cells were grown in LB at 37°C, 240rpm, until an OD ~1. They were subsequently washed and transferred to M9 media with

15

NH4Cl, or

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NH4Cl,

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C6-glucose, and D2O, and

induced with isopropyl β-D-thiogalactopyranoside (IPTG) at 24°C 240 rpm for 20 hours. The cells were harvested by centrifugation at 4000 rpm using a JLA 10.5 rotor for 20 minutes, and then resuspended in 5mM NaH2PO4 at pH 5.85 before freezing overnight at -80°C. The cells were thawed and lysed by adding 5mg lysozyme followed by sonication for 6 minutes (10s on, 50s off). Cellular debris were removed by centrifugation at 17000 rpm for 20 minutes using a JA-20 rotor.

Purification of OXA-24/40 from the supernatant used standard column

chromatography on an ÄKTA FPLC. Briefly, the supernatant was loaded onto a GE HiTrap SP cation-exchange column, pre-equilibrated in lysis buffer. Elution followed a linear gradient to a final concentration of 20 mM NaH2PO4, 500 mM NaCl at pH 7.0. Fractions containing OXA24/40 were then pooled and loaded onto a Sepharose S200 column (GE Healthcare), preequilibrated in 20mM NaH2PO4, and 200mM NaCl at pH 7.0. Generation of P227S variant of OXA-24/40. We generated the mutant construct of P227S OXA-24/40 using the megaprimer PCR method

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in the pET24a plasmid.

The P227S

substitution was confirmed via Sanger Sequencing at the Notre Dame Genomics Facility. We transformed the plasmid into BL21 (DE3) E. coli cells for P227S production.

Protein


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purification followed essentially the same protocol given above for WT OXA-24/40, with only slight changes to the elution time of the mutant peak. Sequential backbone resonance assignments. All NMR samples used buffer containing 20 mM NaH2PO4, 30 mM NaCl, 0.02% NaN3, and 10% D2O at pH 7.0. Spectra were recorded at 16.4 T (700 MHz 1H) on an Avance Bruker system, equipped with a TCI cryoprobe. The nominal sample temperature was 295 K (actual temperature of 290.1 K, determined by a methanol standard). Sequential assignment of backbone NMR resonances used standard double and triple resonance experiments including NH TROSY HSQC 16, 17, and TROSY-based HNCA, HNCO, HN(CA)CO, HNCACB, and HN(CO)CACB

18-20

, followed by analysis with CARA

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.

Protein regions suffering from poor hydrogen back-exchange in partially-deuterated samples were assigned using a

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N-resolved NOESY spectrum in U-15N/1H (protonated) samples.

Assignments of doripenem-acylated OXA-24/40 were determined solely using a NOESY

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N-resolved

spectrum. Assignments of the P227S variant were confirmed using the HNCACB

and HN(CA)CO experiments. NH chemical shift perturbations (CSPs) caused by either the P227S mutation or acylation by doripenem were determined using = ∆ = ∆ + (0.154∆ ) (1)

In eq 1, ∆δH and ∆δ N are chemical shift perturbations for 1HN and

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N, respectively. The

∆δN values are weighted by 0.154, the average ∆δH / ∆δN ratio among chemical shifts recorded in the BMRB database23. 15

N backbone relaxation experiments. To characterize the backbone dynamics of OXA-

24/40 and the P227S variant, we measured standard amide

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N relaxation rate constants,


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including 15N R1 = 1/T1, R2 = 1/T2, and the 15N-1H steady-state NOE 24, 25, incorporating selective pulses to minimize artifacts from NH-solvent proton exchange 26. Measurements were at 16.4 T (700MHz 1H Larmor frequency) and a nominal sample temperature of 295K. The R1 spectra covered relaxation delays of (s) of 0.048 (2X), 0.192, 0.400, 0.592, 0.800, 1.040, 1.344, and 1.552. The R2 spectra included relaxation delays (ms) of 8.24 (2X), 16.48, 24.72, 32.96, 41.20, 49.44, 57.68, and 65.92. The

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N-1H steady-state NOE (ssNOE) experiments were recorded as

interleaved experiments cycling between saturating and non-saturating sequences, per Ferrage et al.

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. The corresponding dipole-dipole cross-relaxation rate constants, σNH, were calculated

from the product ((γN/γH)R1ssNOE). Time-domain data were processed using Topspin 2.1 (Bruker Biospin, Inc.). The NH cross-peaks were integrated and fit to mono-exponential decays using in-house software. Statistical errors were estimated using 512 Monte-Carlo simulations based on integral uncertainties from the duplicate time-point spectra. Reduced Spectral Density Mapping. To extract mobility information about N-H bonds from the 15N-relaxation parameters, we used reduced NH spectral density mapping 28-30. This method produces for each NH bond discrete values of its spectral density of motion, J(ω), directly from linear combinations of the experimental here is that of Peng and Wagner

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N spin relaxation rate constants. The formalism used

. As in our previous studies

31

, we used the effective zero-

frequency spectral density value, Jeff(0), (eq 2),

R 3σ   J eff ( 0 ) = λ  R2 − 1 − NH  (2)  2 5  to profile and compare site-specific NH bond motions. The “λ” coefficient in eq 2 is a ratio of constants, 3/(6D+2C), where C and D are related to the 15N chemical shift anisotropy and 15N-1H dipolar relaxation mechanisms. The λ value is approximately 0.26 (ns/rad)2 at 16.4 T.


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For globular proteins, a plot of Jeff(0) versus NH sequence typically shows a core-average (indicative of overall protein tumbling), punctuated by high and low outlying Jeff(0) values

28, 32

.

These outliers reflect the influence of internal motion on the two distinct terms contributing to Jeff(0), shown in eq 3,

J eff ( 0 ) = J ( 0 ) + λ R ex . (3) The J(0) term is equivalent to a local correlation time for re-orientational NH bond motions relative to the external magnetic field 32. An isotropically tumbling, rigid protein would have the same J(0) for all NH bonds. If overall isotropic tumbling is retained, but conformational flexibility is allowed, then the NH bonds can reorient via additional internal motions on time scales shorter than overall tumbling (i.e. subnanosecond). In particular, NH bonds with large amplitude internal motions on the subnanosecond time scale will show reduced J(0) (and thus, Jeff(0)) relative to the rigid protein case. By contrast, the λRex term increases Jeff(0); this term accounts for the possibility of slower (µs-ms) chemical exchange dynamics that modulate the 15N chemical shift, causing 15N line-broadening, Rex. The λRex term enters through the dependence of Jeff(0) on

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N R2 experiments (eq 2), which sense microsecond-millisecond exchange and

subnanosecond NH bond motions. The dual sensitivities of Jeff(0) to internal motion provided us the means to investigate how the P227S gain-of-activity mutation might perturb WT OXA-24/40 conformational dynamics. The overall shape of OXA-24/40 is only slightly oblate (principal moments of inertia of 1, 1, and 0.7 for PDB entry 3PAE

12

), suggesting that approximation of isotropic overall solution tumbling

was reasonable. Accordingly, we interpreted the Jeff(0) outliers as NH sites with internal motion. Jeff(0) outliers were defined as values outside at least two standard deviations from the core


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average. In particular, NH bonds with low outlying Jeff(0) identified NH bonds with larger amplitude internal motion on the sub-nanosecond time scale. The NH bonds showing high outlying Jeff(0) values identified NH bonds with

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N chemical shifts modulated by micro-

millisecond exchange dynamics. For these high outliers, we estimated the 15N Rex by subtracting the core average J(0) value, and dividing by λ, per eq 3. RESULTS We compared the site-specific backbone dynamics of wild-type (WT) OXA-24/40 with that of the single substitution variant, P227S; the variant gains hydrolase activity against advanced cephalosporins and the monobactam, aztreonam

11

. Comparisons of backbone dynamics during

substrate turnover, while desirable, were complicated by the long duration of the NMR relaxation experiments relative to the time for substrate turnover. Our measurements therefore focused on the apo (unacylated) states of both proteins. Critically, the apo state allowed investigation into two critical and open questions concerning the origins of the expanded substrate activity of P227S: (i) What are the intrinsic (apo-state) backbone dynamics of OXA24/40 in solution?; (ii) How does P227S – a gain of activity mutation – alter those dynamics? Functional residues of OXA-24/40. OXA-24/40 (255 residues, 7 prolines) belongs to the larger superfamily of SxxK bacterial acyl-transferase proteins, which share three conserved motifs: (SxxK), (SxN), and (KS/TG) 33. Figure 1 highlights these motifs in OXA-24/40, which comprise its β-lactam binding pocket and they are crucial for β-lactam acylation and deacylation 12

. The STFK motif (S81-T82-F83-K84, N-terminal side of helix C) contains the serine side

chain hydroxyl (S81) that forms the acyl linkage with the β-lactam. The K84 side chain Nζ is carboxylated, producing a carbamate anion that promotes both acylation and deacylation. The Nζ carboxylation is a post-translational modification characteristic of Class D β-lactamases and the


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extracellular sensor domain of BlaR1 in S. aureus 2. The single site substitution K84D makes OXA-24/40 deacylation deficient

12

. The SAV motif (S128-A129-V130 between helices E and

F) is also essential for deacylation, as demonstrated by the V130D mutation, which also makes OXA-24/40 deacylation-deficient

12

.

Structural insights into the OXA-24/40 acyl-enzyme

complex have come from x-ray structures of both K84D and V130D acylated by doripenem, a member of the carbapenem class of β-lactams 12. Just outside the antibiotic binding pocket are three surface loops that also influence substrate specificity: (i) the P-loop (residues 95-119); (ii) Ω loop (residues 154-174); and (iii) the β5-β6 loop (residues 221-230). Comparisons of x-ray crystal structures of OXA-24/40 10, 14, 34

11-13

, OXA-48

show enhanced local mobility of the β5-β6 loop in the form of higher B-factors and local

structural changes upon substitution mutation. OXA-24/40 shows a distinctive hydrophobic bridge formed by contact between the side chains of P-loop (Y112) and β5-β6 (M223) degrade carbapenemase activity

13

12, 13

. Indeed, the alanine substitutions Y112A, M223A

, suggesting that the bridge promotes proper carbapenem

orientation and longer residence times, resulting in higher binding affinity. The higher affinity would then partially offset the rather slow deacylation rate, resulting in the observed turnover that is sufficient for clinical carbapenem resistance 11. This hydrophobic bridge is not universal: it appears in some CHDLs (e.g. OXA-23

35

) but is lacking in others (e.g. OXA-48

14, 34

). The

bridge may therefore offer an avenue for selective targeting among CHDLs 2. Intrinsic backbone flexibility of WT-OXA-24/40.

Having introduced the functional

segments of OXA-24, we can now describe our NMR studies. We prepared isotope-enriched OXA-24/40 using the same methods described in our recent NMR studies of BlaRS structurally homologous β-lactam sensor domain in the gram-positive S. aureus.

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, a A


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representative 2-D

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N-1H TROSY-HSQC of WT OXA-24/40 at 16.4T, 295K is in the

Supporting Information, Figure S1. We sequentially assigned 223 out of a total of 248 backbone NHs, using standard triple-resonance NMR experiments described previously

31

.

Chemical Shift Index (CSI) 36 analysis of main chain 13Cα/β secondary chemical shifts predicted secondary structure consistent with that evident in the x-ray crystal structures (Supporting Information, Figure S2). Further details concerning sample preparation, sequential assignments, and CSI analysis are in the Materials and Methods. We characterized the flexibility of OXA-24/40 backbone NH bonds by measuring standard 15N relaxation parameters R1 26, R2 37, and heteronuclear steady-state 1HN-15N NOE 38, followed by a reduced spectral density mapping procedure

28

. The results produced for each NH bond a local

mobility parameter, Jeff(0). Figure 2 maps these values onto the OXA-24/40 structure, and a corresponding bar chart is in Figure S3, Supporting Information. The Jeff(0) values gave a core average and standard deviation of 6.83 ± 0.45 ns/rad. High Jeff(0) outliers pinpointed NHs experiencing line broadening due to µs-ms exchange dynamics. Low Jeff(0) outliers pinpointed NH bonds with large amplitude (enhanced) subnanosecond bond motion

28

. Further details

concerning the 15N relaxation experiments and Jeff(0) are in the Materials and Methods. Residues within the three conserved motifs in the antibiotic binding pocket (STFK, SAV, and KSG) showed high outlying Jeff(0) values, indicating Rex line broadening caused by µs-ms exchange dynamics. Notable residues included A126 and V132 bracketing SAV, and K218 and G220 in the KSG motif. Exchange dynamics at these residues suggested a pliable enzyme active site, adaptable to multiple β−lactams with differing side chains. Residues within the peripheral binding loops outside the antibiotic pocket also showed high Jeff(0) outliers: (i) K96, D106, and K108 in the P-loop (95-119); (ii) G222 and V229 in the β5-


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β6 hairpin; (iii) and F166, W167, L168, L172, K173, and I174 in the Ω loop (154-174). Significantly, the P-loop NH resonances of the aforementioned hydrophobic bridge residues are absent (T111-Y112 in the P-loop), and their bracketing residues also show increased Jeff(0) and broadened resonances. These results indicate the P-loop resonances sense local exchange dynamics, and suggest the hydrophobic bridge contact may be a transient rather than static interaction. The trimmed mean and standard deviation of Rex values was 5.6 ± 2.6 s-1. The highest outliers at (> 10 s-1) included K58 in β1; D106 (P-loop); F166, W167, L168 (Ω-loop); K218 (KSG); and F246, L248 (β7 strand). We also observed evidence of NH bonds undergoing faster time scale internal motions (subnanosecond bond vector reorientation), in the form of low Jeff(0) outliers. Such bonds were mainly at residues outside the antibiotic binding pocket. Notable residues included those at the start of the P-loop, as defined by the crystal structure 13 (i.e. H95, T99, I102, and W105) and tip (R110 and M114). The enhanced subnanosecond mobility could reflect local disorder that would promote slower (microsecond-millisecond), more collective P-loop mobility that may account for the P-loop exchange broadening discussed above. Additional sites of large amplitude subnanosecond mobility (low Jeff(0) outliers) included residues at the tip of the β5-β6 loop (T226) and in the neighboring β7-αK loop (K253 and E254). WT OXA-24/40 interactions with doripenem. To investigate the residue-specific effects of acylation, we analyzed the

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N-1H (NH) chemical shift perturbations (CSPs) of 50 µM WT-

OXA-24/40 upon addition of 2.5mM doripenem, a representative carbapenem (Figure 3). CSP magnitudes were quantified according to eq 1 in the Materials and Methods; significant CSPs were those with magnitudes exceeding 0.05 ppm. The NH CSPs persisted for > 48 hours, and ultimately returned to their apo state chemical shifts. This behavior suggested the NH CSPs were


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consequences of OXA-24/40 acylation by doripenem. Further support for this interpretation was our observation of highly similar doripenem-induced NH CSPs in the OXA-24/40 variant, V130D (Supporting Information, Figure S4). Significantly, the V130D variant is the same construct used to solve the crystal structure of OXA-24/40 acylated (covalently bound) by doripenem (PDB ID: 3PAG) 12. Many doripenem-induced CSPs were consistent with contacts relevant for binding antibiotic. For example, the largest CSPs occurred at the three highly conserved motifs that interact with the acylated antibiotic, STFK, SAV, and KSG. There were also large CSPs at the starting and ending terminal residues of the β5-β6 loop (G222, M223, V225, G230, and W231) and P-loop (T99, E116, and T120). These residues are proximal to the loop residues forming the hydrophobic bridge proposed to filter substrate access to the active-site serine 13. Additionally, Ω-loop residues (F166, W167, and L168) near the antibiotic binding site show strong CSPs. This is consistent with observations of van der Waals interactions between L168 and the hydroxylethyl side chain that is characteristic of the carbapenem class of β-lactams 12. In short, the above CSPs were consistent with loop residues involved in formation of the acyl-enzyme complex. At the same time, significant CSPs also occurred at residues lacking direct interaction with doripenem. Examples included Ω-loop residues (I159, L172, K173, and I174); these more distal CSPs may reflect conformational transitions of the Ω-loop to accommodate the bound antibiotic. Other more distal CSPs occurred in the β7-αK turn and αK itself (residues 250-270). Interestingly, these residues lie in a region that is structurally homologous to a cryptic allosteric site identified in TEM-1, a Class A β-lactamase, using Markov-state modeling 39. As stated, the doripenem-induced NH CSPs persisted for > 48 hours, suggesting a long-lived acyl-enzyme complex. Yet, previous steady-state kinetic measurements

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for WT OXA-24/40


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against doripenem predict complete doripenem hydrolysis in < 10 minutes (under VMAX conditions) – a duration much shorter than that used to collect a single 2-D NH TROSY HSQC (~ 1 hour). Thus, the kinetics measurements predict an acyl-enzyme lifetime too short to produce the long-lived doripenem-induced NH CSPs (> 48 hours). We do not yet understand this mismatch in time scales. As described above, the CSP locations in both WT OXA-24/40 and V130D are indicative of a bound complex. The unexpectedly long-lived CSPs may reflect a long-lived non-covalent complex, with doripenem still sequestered in the active site, giving rise to CSPs that overlap with those made in the covalent complex (doripenem acylated OXA-24/40). Alternatively, the long-lived NH CSPs may reflect accumulation of a doripenem-acylated OXA24/40 population with a greatly reduced deacylation rate. The previous kinetic measurements suggesting rapid turnover are based on initial rates, and therefore reflect only the first rounds of turnover 10. Possibly, when OXA-24/40 is challenged with a large excess of doripenem, as in our NMR experiments, OXA-24/40 may convert to a species that stalls in the acyl-enzyme intermediate state.

In fact, the deacylation rate for class D β-lactamases depends on a

carboxylated active site lysine to activate the deacylating water; hence, decarboxylation of the active site lysine might cause this partition to an inactive enzyme OXA-24/40 acyl-doripenem 40

2,

. To investigate these hypotheses, we are assigning active site side chain resonances, and using

alternative methods for rapid 2D spectral acquisition to capture the very early rounds of turnover. P227S mutation alters backbone flexibility of WT OXA-24/40. We generated the P227S mutant of OXA-24/40, which shows broader substrate activity that includes 3rd generation cephalosporins (Ceftazidime and Cefotaxime) and the monobactam Aztreonam

11

. Details

concerning P227S generation, expression, and purification are above in Materials and Methods.


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The 2-D

15

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N-1H TROSY HSQC of P227S was very similar to WT OXA-24/40 (Figure S1,

Supporting Information) indicating overall preservation of the WT fold. consistent with the x-ray crystal structures

11, 12

These results are

, which show 0.165Å RMSD for all Cα atoms

relative to WT OXA-24/40. We compared the P227S NH cross-peak positions relative to WT OXA-24/40 to identify mutation-induced NH CSPs. Most of the CSPs localized to residues at the mutation site (Figure 4). But there were also significant CSPs at residues distal from the mutation site, including the Ω-loop (L168, V169, and G170) and β7-αK loop residues. The CSP of M252 of (β7-αK) may reflect a new hydrogen bond with the side chain hydroxyl of S227. We then used the same

15

N spin relaxation methods described above for WT to determine

Jeff(0) versus sequence for P227S. The core average Jeff(0) of P227S was essentially identical to WT- OXA-24/40 (trimmed mean of 6.77 ± 0.34 ns/rad), indicating retention of overall solution tumbling properties. Hence, we could map changes in site-specific backbone dynamics by evaluating the simple difference of P227S versus WT Jeff(0) values along the backbone. Indeed, P227S showed local differences in Jeff(0) relative to WT OXA-24/40, as indicated in Figure 5 (Corresponding bar chart in Figure S5, Supporting Information). These differences occurred at NH bonds that had shown evidence of significant local dynamics in WT OXA-24/40. For example, P227S showed marked changes at NH bonds in β5-β6 and the P-loop that had shown evidence of large amplitude internal motion on the subnanosecond time scale in WT OXA-24/40 (i.e. low outliers in Jeff(0)). The β5-β6 residues included G224 and T226, next to the PS substitution site, while the P-loop residues were T99, R110, and M114. In WT, the NH bonds of these residues showed Jeff(0) significantly less than the core average, whereas in P227S, their values were much closer to the core average.


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P227S also showed differences on the microsecond-millisecond time scale. In particular, P227S showed marked changes altered at NH bonds whose WT Jeff(0) values were high outliers and thus, sites indicative of exchange dynamics. These altered NH bonds included the conserved motif residues within the antibiotic binding pocket, SAV (M125, A126, L127, and V132) and STFK (T82), as well as the Ω-loop (Q162, F166, W167, L168, and I174). In WT, the Jeff(0) values of these NH bonds were high outliers. By contrast, in P227S, their values were much closer to the core average. For P227S, the trimmed average and standard deviation of the Rex was (4.1 ± 1.0 s-1) versus WT OXA-24/40 (5.6 ± 2.6 s-1). The largest values were at D106 (Ploop), K150, and L168 (Ω-loop). Overall, P227S reduced the extent of microsecond-millisecond exchange dynamics seen in WT OXA-24/40. The reduction could reflect mutation-induced changes in 15N chemical shifts, exchange-coupled populations, or exchange rate constants. While the underlying causes are unclear, it is worth noting that the Cα-Cα distances between P227 in the β5-β6 loop and T82 in the conserved STFK segment are >15 Å; hence, their coincident changes in Jeff(0) suggest side-chain interactions coupling these two important functional sites. In summary, the P227S substitution perturbed WT backbone flexibility in segments important for β-lactamase activity, enhancing the flexibility in some segments (e.g. β5-β6), reducing it in others (e.g. Ω-loop, SAV and STFK within the antibiotic binding pocket). These dynamic perturbations must be considered when trying to explain the gain in P227S cephalosporinase activity, and the decrease in KM (vide infra). P227S OXA-24/40 binds doripenem similarly to wild-type OXA-24/40. We then investigated the effects of acylation by addition of 2.5 mM of doripenem to 50 µM P227S OXA24/40 and calculating the 15N-1H CSPs. We used the same concentrations as in our studies of WT described above. The doripenem-induced CSPs in P227S closely matched those of WT; the


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majority of the changes in CSPs were less than the cut-off (0.05 ppm) used to identify significant doripenem-induced CSPs in WT-OXA-24/40 (Figure S6, Supporting Information). The close match suggested overall preservation of the WT binding mode and affinity. The broader substrate activity of P227S compared to WT manifests as the former’s ability to bind and hydrolyze advanced cephalosporins (e.g. ceftazidime) 11. We therefore explored P227S CSPs upon addition of ceftazidime. The results indicated substantially weaker binding relative to doripenem. We needed a 350-fold excess of ceftazidime (35 mM ceftazidime versus 100 µM P227S) to observe CSPs. The large molar excess of antibiotic produced t1-ridges in our NMR spectra that precluded analysis of some 15N-1H cross-peaks of P227S. Nevertheless, the majority of residues that showed ceftazidime-induced CSPs also showed doripenem-induced CSPs, albeit often in different magnitudes and directions. An example is the well-isolated peak of G222 at the start of the β5-β6 loop (Figure S7, Supporting Information). In P227S, the ceftazidimeinduced CSP in P227S differs in direction (sign) from the doripenem-induced CSP in both P227S and WT OXA-24/40. The directional difference may reflect the different side chain substituents of ceftazidime versus doripenem, a different binding mode, or both.

DISCUSSION Gain-of-activity mutations among carbapenem-hydrolyzing Class D β-lactamase (CHDLs) are expanding β-lactam resistance in gram-negative pathogens 2, 10, 41. Here, we present NMR studies to better understand the origins of these gain-of-activity mutations. Our studies investigate OXA24/40, a CHDL responsible for β-lactam resistance in A. baumannii, and one of its clinical variants, P227S. Like WT, P227S is active against the carbapenems and penicillins. But unlike


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WT, P227S is also active against advanced cephalosporins and the monobactam, aztreonam

11

.

Hence, P227S shows expanded β-lactam substrate activity compared to WT OXA-24/40. Initial explanations for the expanded activity of P227S have come from x-ray crystal structures 12, 41

, which include P227S acylated with the new substrate, ceftazidime

11

acylated with its natural substrate, doripenem (a representative carbapenem)

, and OXA-24/40 12

. The structures

show that neither the WT residue P227 nor the variant residue S227 directly contacts the acylated antibiotic. Instead, in P227S, the β5-β6 loop adopts a new configuration that is distinct from that observed in WT OXA-24/40-doripenem structure. Significantly, the new configuration can bind ceftazidime but not doripenem, because the β5-β6 loop moves away from the catalytic serine to expand the binding pocket. The inactivity of WT OXA-24/40 against ceftazidime suggests that its β5-β6 sequence cannot easily access the new ceftazidime-compatible configuration adopted by P227S. These previously noted changes in local β5-β6 structure indicated enhanced local flexibility, as do other crystal structures of related CHDLs (higher B-factors, alternate loop conformations upon substitution mutations)

10, 14, 34, 42

. The coincidence of expanded substrate activity with

perturbations of β5-β6 conformation suggested that mutations of the β5-β6 loop sequence might alter the breadth of local conformations relevant for β-lactam substrate recognition. OXA-24/40 function mediated by conserved, flexible motifs. We investigated this possibility by using NMR to compare the site-specific backbone dynamics of WT OXA-24/40 and the P227S variant. To our knowledge, this is the first study of CHDL conformational dynamics by NMR, and its role in contributing to antibiotic resistance. Our results show unequivocally that the OXA-24/40 segments important for substrate activity are intrinsically flexible over a broad time scale. These segments include the conserved STFK,


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SAV, and KSG motifs within the antibiotic binding pocket, and the peripheral loops “guarding” entry to the pocket. Our results therefore strengthen the notion that an understanding of the OXA-24/40 mechanism requires study of its dynamic properties alongside its chemical and structural properties. Single site substitutions expanding the OXA-24/40 substrate profile alter the mobility of conserved motifs. The recent P227S crystal structures

11

showed a pronounced shift of the β5-

β6 loop away from the antibiotic binding pocket, making it possible to accommodate new substrates with bulkier side chains, such as the advanced cephalosporin, ceftazidime. P227S also showed higher enzyme efficiency (increased kcat/KM) than WT against ceftazidime and Aztreonam, primarily by reduced KM

11

. The reduced KM of P227S could translate into higher

rates of antibiotic turnover, particularly at low substrate concentrations. Our NMR results provide the first insights into the dynamical consequences of this activityexpanding mutation. In particular, P227S alters the backbone flexibility profile of WT OXA24/40 on both the microsecond-millisecond and sub-nanosecond time scales. These occur at conserved residue motifs defining the antibiotic binding pocket, and the peripheral β5-β6 and Ω loops that modulate substrate specificity. P227S has lost most of the high outlying Jeff(0) values in the SAV region and the Ω-loop that had been prominent in WT OXA-24/40.

In P227S, they are closer to core average. This

indicates a loss of WT µs-ms exchange dynamics and consequent 15N line broadening. This effect could reflect a reduction of conformational exchange within the antibiotic binding pocket, possibly enhancing active site pre-organization for more facile substrate acylation. Such preorganization could contribute to the reduction in KM. In considering possible influences on KM, we are mindful that KM is not KS, the substrate equilibrium dissociation constant. Rather, the KM


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value generally depends on rate constants for turnover, binding, or both (Supporting Information, Figure S8). Mutations lowering KM, such as P227S, may reflect perturbations to multiple rate constants. Only in the limit that acylation is much slower than substrate dissociation from the enzyme, KM then becomes a measure of KS.

In this limiting case, the reduced

conformational mobility of the P227S antibiotic binding pocket could reflect increased stability of the Michaelis complexes, leading to smaller koff, and thence, KM. This remains speculative, as the relative time scales for acylation versus substrate dissociation have not yet been established. P227S also shows evidence of larger amplitude NH bond motions on the subnanosecond time scale (decreased Jeff(0) values) in the β5-β6 substrate recognition loop, relative to WT. The increased flexibility would be consistent with broader conformational sampling by the β5-β6 loop to promote broader substrate recognition and activity for P227S relative to WT, including advanced cephalosporins. The enhanced loop flexibility could also increase the substrate association rate constant kON, thus reducing KM, but only in the special case that KM is a proxy for KS (vide supra). Similar binding loop flexibility changes resulting in enhanced substrate spectrum have been recently reported for the Bacillus cerus metallo-B-lactamase II (BcII) 43, 44. Hypotheses regarding the effect of P227S on β5-β β6 conformations. P227S in the β5-β6 loop broadens the original WT OXA-24/40 substrate spectrum of penicillins and carbapenems to further include advanced cephalosporins. Our NMR studies above show P227S also perturbs β5β6 conformational mobility, relative to WT. These results stimulate two hypothetical mechanisms linking these observations. In the first mechanism, P227S could allow β5-β6 to sample a new set of conformations inaccessible to WT OXA-24/40. The new set would be capable of binding both carbapenems


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(e.g. doripenem) and advanced cephalosporins (e.g. ceftazidime). This new set would be expected to bind doripenem differently from WT OXA-24/40. In the second mechanism, P227S may simply broaden the range of β5-β6 conformational sampling. This range would retain the WT-doripenem conformation, but now include additional conformations with more facile access to advanced cephalosporins. The great similarity of doripenem-induced CSPs between WT and P227S (Figure S6, Supporting Information), along with the different CSPs of G222 for P227S (Figure S7, Supporting Information) in complex with doripenem versus ceftazidime strongly favors the second mechanism. The difference between the G222 CSPs for the two P227S complexes may reflect interactions with the differing side chains of doripenem versus ceftazidime.

But these

differences may also reflect the differences in local backbone conformation needed to accommodate the distinct antibiotics. Amide

15

N and 1HN chemical shifts are sensitive to local

backbone torsions Ψi-1 and Φi, as well as ring current effects (such as those from nearby aromatic side chains W221), and hydrogen-bonding 45. In this context, we note that G222 Φ angle shows considerable changes between the OXA-24/40 and P227S in the crystal structures

11

. Further

support comes from the pattern of dynamic change in Figure 5 above. Specifically, residues at the tip of β5-β6 show an increase in subnanosecond re-orientational motions, which could allow access to alternate conformations as seen in the ceftazidime bound complex.

CONCLUSION We have investigated the conformational dynamics of OXA-24/40 and single substitution mutant, P227S from a clinical strain of A. baumannii showing broader substrate activity. Our NMR results provide the first glimpses at the per residue flexibility of a clinically relevant


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CHDL and its response to mutations causing enhanced antibiotic resistance. In particular, they provide new insight to complement the existing crystal structures, and now enable dual consideration of both structure and dynamics when trying to understand how protein function evolves under selective drug pressure. We find that conserved segments of OXA-24/40 involved in substrate activity and specificity are intrinsically flexible on both the sub-nanosecond and micro-millisecond time scales. Moreover, the P227S mutation alters the flexibility of those segments, enhancing the conformational flexibility of some parts, while reducing in others, with the net effect of expanding the breadth of β-lactams it can bind and hydrolyze. These findings take on broader significance in light of our recent studies of the β-lactam sensor domain BlaRS 31, 46, which binds β-lactam antibiotics on the cell surface, initiating trans-membrane signal transduction to stimulate production of β-lactamases in the gram-positive pathogen, Methicillin-resistant Staphylococcus aureus (MRSA). Despite only ~ 26 % sequence identity, BlaRS and OXA-24/40 folds are quite similar, (0.9 Å Cα RMSD, 148 Cα aligned atoms). In BlaRS, we showed that the homologous β5-β6 loop also harbors functional dynamics that are altered by antibiotic binding; there, however, the BlaRS loop fluctuations appear to promote trans-membrane signal transduction

31, 46

. Taken together, our results suggest that the functional β5-β6 loop motion in

OXA-24/40 may be a general feature of the CHDL architecture. They further suggest that enzyme evolution varies not only familiar properties such as chemical groups and structure, but also intrinsic conformational dynamics. If so, then we should take seriously the notion that antibiotic drug resistance can emerge from a variety of mechanisms, including perturbations of sequence-dependent functional dynamics. In particular, more aggressive efforts to correlate


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sequence-dependent protein dynamics with substrate specificity could enhance predictions of CHDL mutations expanding antibiotic resistance. ASSOCIATED CONTENT Supporting Information. Supporting figures S1-S8. NH-TROSY spectra of wild-type OXA24/40 and P227S (Figure S1), Chemical Shift Index analysis of wild-type OXA-24/40 (Figure S2), graph of Jeff(0) values for WT OXA-24/40 (Figure S3), comparison of CSPs for WT and V130D OXA-24/40 in complex with doripenem (Figure S4), graph of ∆ Jeff(0) values for P227SWT OXA-24/40 (Figure S5), graph of changes in CSPs for WT and P227S OXA-24/40 in complex with doripenem (Figure S6), NH spectra of G222 of P227S in complex with doripenem and ceftazidime (Figure S7), and a scheme of the hydrolytic mechanism with rate constants contributing to KM and kcat for β-lactam hydrolysis (Figure S8).

AUTHOR INFORMATION Corresponding Author * Jeffrey W. Peng, [email protected]. ACKNOWLEDGMENT We thank Dr. Thomas E. Frederick, Mr. Brendan J. Mahoney, and Mr. Heath A. Rose for valuable discussions. References [1] Papp-Wallace, K. M., Endimiani, A., Taracila, M. A., and Bonomo, R. A. (2011) Carbapenems: past, present, and future, Antimicrob Agents Chemother 55, 4943-4960. [2] Leonard, D. A., Bonomo, R. A., and Powers, R. A. (2013) Class D beta-lactamases: a reappraisal after five decades, Acc Chem Res 46, 2407-2415. [3] Bou, G., Oliver, A., and Martinez-Beltran, J. (2000) OXA-24, a novel class D beta-lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain, Antimicrob Agents Chemother 44, 1556-1561. [4] Poirel, L., Naas, T., and Nordmann, P. (2010) Diversity, epidemiology, and genetics of class D beta-lactamases, Antimicrob Agents Chemother 54, 24-38. [5] Peleg, A. Y., Seifert, H., and Paterson, D. L. (2008) Acinetobacter baumannii: emergence of a successful pathogen, Clin Microbiol Rev 21, 538-582.


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[36] Wishart, D. S., and Sykes, B. D. (1994) The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data, J Biomol NMR 4, 171-180. [37] Kay, L. E., Nicholson, L. K., Delaglio, F., Bax, A., and Torchia, D. A. (1992) Pulse Sequences for Removal of the Effects of Cross Correlation betwen Dipolar and Chemical-Shift Anisotropy Relaxation Mechanisms on the Measurement of Heteronuclear T1 and T2 Values in Proteins, J. Magn. Reson. 97, 359-375. [38] Ferrage, F., Cowburn, D., and Ghose, R. (2009) Accurate sampling of high-frequency motions in proteins by steady-state (15)N-{(1)H} nuclear Overhauser effect measurements in the presence of cross-correlated relaxation, J Am Chem Soc 131, 60486049. [39] Bowman, G. R., Bolin, E. R., Hart, K. M., Maguire, B. C., and Marqusee, S. (2015) Discovery of multiple hidden allosteric sites by combining Markov state models and experiments, Proc Natl Acad Sci U S A 112, 2734-2739. [40] Golemi, D., Maveyraud, L., Vakulenko, S., Samama, J. P., and Mobashery, S. (2001) Critical involvement of a carbamylated lysine in catalytic function of class D betalactamases, Proc Natl Acad Sci U S A 98, 14280-14285. [41] Mitchell, J. M., and Leonard, D. A. (2014) Common clinical substitutions enhance the carbapenemase activity of OXA-51-like class D beta-lactamases from Acinetobacter spp, Antimicrob Agents Chemother 58, 7015-7016. [42] June, C. M., Vallier, B. C., Bonomo, R. A., Leonard, D. A., and Powers, R. A. (2014) Structural origins of oxacillinase specificity in class D beta-lactamases, Antimicrob Agents Chemother 58, 333-341. [43] Gonzalez, M. M., Abriata, L. A., Tomatis, P. E., and Vila, A. J. (2016) Optimization of Conformational Dynamics in an Epistatic Evolutionary Trajectory, Mol Biol Evol 33, 1768-1776. [44] Meini, M. R., Tomatis, P. E., Weinreich, D. M., and Vila, A. J. (2015) Quantitative Description of a Protein Fitness Landscape Based on Molecular Features, Mol Biol Evol 32, 1774-1787. [45] Han, B., Liu, Y., Ginzinger, S. W., and Wishart, D. S. (2011) SHIFTX2: significantly improved protein chemical shift prediction, J Biomol NMR 50, 43-57. [46] Frederick, T. E., Wilson, B. D., Cha, J., Mobashery, S., and Peng, J. W. (2014) Revealing cell-surface intramolecular interactions in the BlaR1 protein of methicillin-resistant Staphylococcus aureus by NMR spectroscopy, Biochemistry 53, 10-12.

FIGURE LEGENDS

Figure. 1 Conserved active site segments within OXA-24/40 (PDB 3PAE 12): cyan, STFK (8184), light green SAV (128-130), yellow KSG (218-220). Binding loops outside the active site include the P-loop (95-119) in purple, Ω-loop (154-174) in orange, and β5-β6 loop in dark green.


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Figure 2. Intrinsic backbone flexibility of wild-type OXA-24/40 mapped by Jeff(0) values. Blue spheres are residues with enhanced sub-nanosecond re-orientational motions of NH bonds. Red spheres are 15N sites experiencing line-broadening due to µs-ms exchange dynamics.

Figure. 3 OXA-24/40 NH (CSPs) induced by doripenem. (A) Blue bars highlight significant CSPs (trimmed mean 0.017 ppm + 2 standard deviations 0.015 ppm). Cyan, light green, and yellow boxes correspond to the conserved STFK, SAV, and KSG motifs, respectively. (B) Blue spheres are NHs with significant CSPs shown as blue bars in (A).

Figure. 4. OXA-24/40 NH CSPs due to the P227S mutation. (A) Blue bars indicate residues with significant CSPs (trimmed mean 0.017 ppm + 2 standard deviations 0.015 ppm). Cyan, light green, and yellow boxes correspond to the conserved STFK, SAV, and KSG motifs, respectively. (B) Blue spheres are NHs with significant CSPs shown as blue bars in (A). Figure. 5 Changes in backbone NH flexibility upon P227S substitution. Differences J (0) J (0) are plotted on the structure. Blue spheres are residues with a decrease of Jeff(0) more than 0.5 ns/rad (2.5 s.d.), indicating decreased exchange broadening, increased amplitude of subnanosecond mobility, or both. Red spheres are residues with an increase of Jeff(0) > 0.5 ns/rad.


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Biochemistry

FIGURES Figure 1


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Figure 2


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Figure 3 A

B


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Figure 4 A

B


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Figure 5


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ToC Figure


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40 in Carbapenem-Resistant

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