Structural basis of rifampin inactivation by rifampin

Xiaofeng Qia,b, Wei Lina,1, Miaolian Maa, Chengyuan Wanga,b, Yang Hea,b, Nisha Heb,c, Jing Gaod, Hu Zhoud, Youli Xiaoc, Yong Wangc, and Peng Zhanga,2

aNational Key Laboratory of Plant Molecular Genetics, Chinese Academy of Sciences Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; bUniversity of Chinese Academy of Sciences, Beijing 100039, China; cChinese Academy of Sciences Key Laboratory of Synthetic Biology, Chinese Academy of Sciences Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; and dChinese Academy of Sciences Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

Edited by Alexander Serganov, New York University, New York, NY, and accepted by the Editorial Board March 1, 2016 (received for review November 30, 2015) Rifampin (RIF) is a first-line drug used for the treatment of the target, the RNAP β-subunit; these mutations significantly de- tuberculosis and other bacterial infections. Various RIF resistance crease the binding of and thus neutralize the antibiotic mechanisms have been reported, and recently an RIF-inactivation activity (10). Another prevalent resistance strategy adopted by , RIF phosphotransferase (RPH), was reported to phosphor- is modification of the rifamycins, such as ADP ribosy- ylate RIF at its C21 hydroxyl at the cost of ATP. However, the lation, glycosylation, and phosphorylation (11–13). These covalent underlying molecular mechanism remained unknown. Here, we modifications occur on the critical hydroxyls of the 1-amino, solve the structures of RPH from Listeria monocytogenes (LmRPH) 2-naphthol, 4-sulfonic acid (ansa) chain of rifamycins and in different conformations. LmRPH comprises three domains: an thus make rifamycins unable to fit into the binding pocket on ATP-binding domain (AD), an RIF-binding domain (RD), and a cat- RNAP. Additional resistance mechanisms have been reported alytic His-containing domain (HD). Structural analyses reveal that also (14–16). the C-terminal HD can swing between the AD and RD, like a toggle Antibiotic resistance is a great threat to the treatment of in- switch, to transfer phosphate. In addition to its catalytic role, the fectious disease, and understanding the molecular mechanisms HD can bind to the AD and induce conformational changes that of resistance no doubt will help guide the development of a new stabilize ATP binding, and the binding of the HD to the RD is re- generation of drugs (17, 18). A number of studies have been quired for the formation of the RIF-binding pocket. A line of hy- carried out to understand resistance caused by RNAP drophobic residues forms the RIF-binding pocket and interacts mutations (1, 19). However, the and mechanisms in- with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moie- volved in the covalent modifications of rifamycins remain largely ties of RIF. The R group of RIF points toward the outside of the unknown. Recently, an antibiotic-resistance family, RIF pocket, explaining the low substrate selectivity of RPH. Four resi-

phosphotransferase (RPH), was found to inactivate RIF by BIOCHEMISTRY dues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and phosphorylating it at the hydroxyl attached to the C21 of its ansa Gln337, were found to play essential roles in the phosphorylation chain. RPHs in heterologous bacteria are able to inactivate diverse of RIF; among these the His825 residue may function as the phos- phate acceptor and donor. Our study reveals the molecular mech- Significance anism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins. Rifampin (RPH) belong to a recently iden- tified antibiotic-resistance that inactivates rifam- antibiotic resistance | rifampin | phosphotransferase | pin, the first-line drug against tuberculosis, by phosphorylation. molecular mechanism | toggle switch By determining the structures of RPH from Listeria mono- cytogenes (LmRPH) in different conformations, we reveal a ifamycins are a group of natural or semisynthetic antibiotics toggle-switch mechanism of the LmRPH catalytic process in Rused for treating a broad repertoire of bacterial infections. which the C-terminal His domain swings between the ATP- β These compounds bind directly to the -subunit of bacterial binding domain and the rifampin-binding domain to transfer RNA (RNAP) at a highly conserved region, blocking phosphate from ATP to rifampin. These structures explain the the exit tunnel for RNA elongation and thus inhibiting the process low substrate selectivity of RPH for the rifamycins. The residues of (1). The first member of the rifamycins to be de- involved in rifampin phosphorylation are identified also. The scribed, rifamycin B, was extracted from the soil actinomycete structural mechanism revealed in this study will guide the de- Amycolatopsis mediterranei (2). The natural product had modest velopment of a new generation of rifamycins. antibiotic activity, but semisynthetic derivatives of the rifamycin family have proven highly successful in the clinic (3). The best-known Author contributions: X.Q. and P.Z. designed research; X.Q., W.L., M.M., C.W., Y.H., N.H., member of the rifamycin family, rifampin (RIF), was introduced to J.G., and H.Z. performed research; X.Q., W.L., M.M., C.W., Y.H., N.H., J.G., H.Z., Y.X., Y.W., the clinic in 1968; it is highly effective against Mycobacterium tuber- and P.Z. analyzed data; and P.Z. wrote the paper. culosis and greatly shortens the duration of tuberculosis therapy (4). The authors declare no conflict of interest. At present, RIF continues to be a first-line drug for the treatment of This article is a PNAS Direct Submission. A.S. is a guest editor invited by the Editorial tuberculosis (5). Through the years additional derivatives have Board. been developed to treat a wider range of bacterial infections (3); Data deposition: The structural factors and coordinates reported in this paper have been deposited in the (PDB) [PDB ID codes 5HV1 (LmRPH–ANP–RIF), 5HV2 for example, rifalazil serves as an effective antibiotic against (LmRPHG527Y–apo), 5HV3 (LmRPHG527Y–ANP), and 5HV6 (LmRPH–AD)]. Chlamydia-based persistent infections (6), and is used 1Present address: Waksman Institute of Microbiology, Rutgers, The State University of to treat travelers’ diarrhea and irritable bowel syndrome (7, 8). New Jersey, Piscataway, NJ 08854. Extensive use of rifamycins has led to the development of 2To whom correspondence should be addressed. Email: [email protected]. bacterial resistances (9). In M. tuberculosis and other mycobacteria This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the most common resistance mechanisms are point mutations of 1073/pnas.1523614113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1523614113 PNAS | April 5, 2016 | vol. 113 | no. 14 | 3803–3808 Downloaded by guest on September 28, 2021 at the cost of ATP and confers the bacteria with high-level re- sistance to RIF.

Structures of LmRPH at Different Conformations. The LmRPH protein was purified further using gel filtration before crystalli- zation, and the two major peaks (peaks 1 and 2) observed were both confirmed to be LmRPH proteins with similar molecular radius/mass by dynamic light scattering (DLS) (Fig. S2). This result indicates that LmRPH might have different conformations in solution. However, we could obtain diffractable crystals only with LmRPH protein from peak 1. The LmRPH structure was + solved in an AMP–PNP (ANP)-, Mg2 - and RIF-bound state (LmRPH–ANP–RIF) by the single-wavelength anomalous dispersion (SAD) method. The overall structure adopts a saddle-like shape, with the AD (residues 1–315) and the RD (residues 323–748) forming two flaps of the saddle. The C-terminal HD (residues 771–867) binds to theRDfromtheconcavesideofthe“saddle” (Fig. 2A). The ATP analog, ANP, binds in a cleft of the AD from the concave side of the saddle, and RIF binds in a pocket of the RD from the convex side. The two substrate-binding sites are about 49 Å apart, leaving ample room for the HD to play an indispensable role in catalysis. In- triguingly, the HD is linked to the RD by a long, flexible linker – Fig. 1. Activity of LmRPH. (A) In vitro LmRPH reaction products analyzed by (residues 749 770) through which the HD might swing between HPLC with the RIF detection program. (B) Identification of the two peaks in A the AD and RD to transfer phosphate from ATP to RIF. by LC-MS. (Left) Peak 1. (Right) Peak 2. (C) The samples in A were analyzed In the LmRPH–ANP–RIF structure, the HD contacts the RD by HPLC with a -detection program. (D) E. coli growth assay. mainly through hydrophobic interactions (Fig. S3). We in- Bacteria transformed with LmRPH or vector were cultured in solid LB me- troduced mutations at this interface to disrupt these interactions dium complemented with 0, 10, 100, or 1,000 μg/mL RIF. and found that LmRPH proteins containing these mutations had gel-filtration profiles different from those of wild-type proteins (Fig. 2B), i.e., two major peaks for wild-type proteins vs. one clinically used rifamycins with great efficiency (13). Bioinformatic major peak for mutants. Accordingly, LmRPH-G527A, LmRPH- analyses suggest that RPHs are widespread in both pathogenic G527S, and LmRPH-G527Y mutants have much decreased or and nonpathogenic bacteria. The RPH protein contains three no RIF-phosphorylation activity in vitro and reduced or no RIF domains (listed from the N terminus to the C terminus): the resistance in vivo (Fig. 2 C and D). These data suggest that, in- ATP-binding domain (AD), the RIF-binding domain (RD), and stead of two conformations, the LmRPH-G527A, LmRPH- the His domain (HD), which contains a conserved His residue G527S, and LmRPH-G527Y mutants tend to adopt one con- essential for phosphate transfer. This architecture is similar to formation in solution. Indeed, the structures of LmRPHG527Y in that of phosphoenolpyruvate (PEP) synthase, which also contains both the apo form (LmRPHG527Y–apo) and the ANP-bound three domains, an ATP-binding domain, a catalytic His domain, and form (LmRPHG527Y–ANP) were in a conformation different a pyruvate-binding domain and catalyzes the reversible conversion from that of the LmRPH–ANP–RIF structure (Fig. 2 E and F), of ATP, water, and pyruvate to AMP, inorganic phosphate (Pi), and with the HD binding to the AD from the concave side. Notably, PEP (20). Apart from this information, little is known about RPHs. even though the interactions between the HD and the RD and Here we report the crystal structures of RPH from Listeria between the HD and the AD have been observed in different monocytogenes (LmRPH) in different catalytic conformations. LmRPH conformations, we could not quantify the interaction Structural and functional analyses reveal the molecular basis of affinities between the individually purified HD and RD or AD in substrate binding, phosphate transfer, and RIF phosphorylation isothermal titration calorimetry (ITC) experiments; the interac- by LmRPH. This study identifies the molecular mechanism of tions are too weak to be detected by ITC (Fig. S4), suggesting RIF phosphorylation and will guide strategies to overcome RPH- that the interactions between the HD and the RD and between mediated rifamycin resistance. the HD and the AD are dynamic. Our structural data demon- strate that the HD can swing between the RD and the AD, as is Results required for LmRPH catalysis, more specifically, for the transfer Characterization of LmRPH. The encoding RPH from of phosphate from ATP to RIF. LmRPH was cloned, expressed in , and purified. The enzymatic activity of the recombinant LmRPH was tested in ATP Binding with the AD Is Stabilized by the HD. Although the a reaction system containing the substrates RIF and ATP, and LmRPHG527Y–ANP and LmRPH–ANP–RIF structures adopt the products were separated by HPLC. As the reaction proceeded, different conformations, both can bind with ANP, prompting us the amount of RIF gradually decreased, accompanied by the in- to determine their ATP-binding affinities. The results show that G527Y crease of a subsequent product peak (Fig. 1A), which was identi- the ATP-binding affinity of LmRPH (Kd = 0.43 μM) is fied as phosphorylated RIF (RIF-P) by LC-MS (Fig. 1B and Fig. higher than that of the wild-type protein (Kd = 1.11 μM), but the S1). The other substrate, ATP, was converted into AMP rather separated AD itself cannot bind with ATP (the affinity is too low than ADP until RIF was phosphorylated completely (Fig. 1C). to be detected by ITC) (Fig. 3A), suggesting that the HD may To examine ability of LmRPH to inactivate RIF in vivo, E. coli contribute to the ATP binding. To resolve this mystery, we solved BL21 (DE3) cells transformed with pQE80L-LmRPH were cul- the structure of the AD in apo state (LmRPH–AD) and compared tured on solid LB medium containing a gradient of RIF concen- it with the LmRPHG527Y–apo structure (Fig. 3B). The AD contains trations. The results show that E. coli growth is strongly inhibited by two subdomains, subdomain I (residues 1–183) and subdomain II 10 μg/mL RIF, but the introduction of LmRPH at concentrations (residues 190–315), which are connected by a flexible linker (L13, greater than 1,000 μg/mL confers resistance to RIF (Fig. 1D). These residues 184–189) to form a hinge-like conformation. The binding data suggest that LmRPH catalyzes the conversion of RIF to RIF-P of the HD with the AD induces significant conformational changes

3804 | www.pnas.org/cgi/doi/10.1073/pnas.1523614113 Qi et al. Downloaded by guest on September 28, 2021 BIOCHEMISTRY

+ Fig. 2. Overall structures of LmRPH at different states. (A) Structure of wild-type LmRPH in complex with RIF, ANP, and Mg2 . The AD, RD, and HD are colored + lemon, light blue, and orange, respectively. ANP and RIF are shown as sticks and are colored green and magenta, respectively. Mg2 is shown as a sphere. (B) Gel-filtration profiles of wild-type LmRPH (red) and the G527A (green), G527S (cyan), and G527Y (blue) mutants. (C) In vitro catalytic activity of Gly527 mutants detected by HPLC. The reaction time of G527A, G527S, and G527Y is 1 h, and that of wild-type LmRPH is 5 min. Proteins were used at 0.5 mg/mL. (D) E. coli growth assay for Gly527 mutants. Bacteria transformed with wild-type LmRPH, vector, G527A, G527S, or G527Y were cultured in solid LB medium + complemented with 0, 10, 40, or 80 μg/mL RIF. (E) Structure of LmRPHG527Y in apo form. (F) Structure of LmRPHG527Y in complex with ANP and Mg2 . Color codes in E and F are as A.

in both subdomain I and II: helices α4, α5, and α8 of subdomain I (residues 123–134) from subdomain I, which is disordered in the undergo a dramatic shift toward the HD, leading to hydrophobic absence of ANP, can be seen clearly after ANP binding. ANP interactions between α8 (subdomain I) and α31 (HD), and helix binding also induces a conversion of the α9 from subdomain II. α9 unwinds to bind with the HD, also through hydrophobic inter- The formation of L9 and α9 after ANP binding generates steric actions (Fig. 3B). As a result, the conformations of subdomains I repulsions of the HD, thereby weakening the interaction be- and II of the AD are stabilized by the binding of the HD, as is the tween the AD and at HD, as reflected by the poor electron ATP-binding cleft between these two subdomains. These findings density of the HD in the LmRPHG527Y–ANP structure (Fig. S5). explain why the HD is required for tight binding of ATP. In The structural rearrangements of the AD described above the LmRPHG527Y mutant, the HD is restricted from binding with accommodate the tight binding of ANP to the cleft through a + the RD; therefore the binding affinity of LmRPHG527Y is higher number of conserved residues in addition to an Mg2 (Fig. 3D). than that of the wild-type protein (Fig. 3A). Specifically, the adenine ring of ANP forms three hydrogen The binding of ANP to the cleft of the AD results in further bonds with the guanidine group of Arg117, the carbonyl oxygen conformational changes in the surrounding structural elements, of Gln184, and the side chain of Gln183; the 2′-hydroxyl group of as can be seen clearly by comparing the LmRPHG527Y–apo and the ANP ribose forms a hydrogen bond with the side chain of LmRPHG527Y–ANP structures (Fig. 3C). After ANP binding, Glu297; the α, γ-phosphates of ANP form hydrogen-bonding β3–β4, which adopts a loop conformation in the LmRPHG527Y– interactions with residues Arg117, Thr136, Lys22, Arg311, and apo structure, forms a five-stranded antiparallel β-sheet with Gly132; and the β, γ-phosphates of ANP are coordinated with + β1–β2–β5, as do β10–β11 in subdomain II. In addition, loop L9 residues Glu297 and Gln309 through the Mg2 . The importance

Qi et al. PNAS | April 5, 2016 | vol. 113 | no. 14 | 3805 Downloaded by guest on September 28, 2021 Fig. 3. ATP-. (A) ATP-binding affinity of the AD (green isotherm), wild-type LmRPH (blue isotherm), and the G527Y mutant (red isotherm) measured by ITC. (B) Conformational changes of the AD induced by HD binding. The LmRPH–AD struc- ture (gray) is superposed with the AD of the LmRPHG527Y–apo structure (lemon). The L13 loop connecting subdomains I and II is highlighted in red. The interaction interfaces between the AD and HD (orange) are shown in zoom-in views, and residues constituting the interface are shown with side chains. (C) Conformational changes induced in the AD and HD by ANP binding. The AD (lemon) and HD (orange) of the LmRPHG527Y–apo structure are superposed with those of the LmRPHG527Y-ANP structure (light blue). Structural elements undergoing conforma- tional changes after ANP binding are colored in red. (D) Residues constituting the ATP-binding site. ANP (green) and residues (lemon) are shown as sticks, + and Mg2 is shown as a lemon sphere. Coordination and hydrogen bonds are shown as dashed lines. (E) In vitro catalytic activity of ATP-binding site mutants detected by HPLC. The amounts of en- zymes used in the assays of the K22A, R117A, E297A, T136A, Q309A, and R311A mutants are 10× those used in assays of wild-type LmRPH. (F) E. coli growth assay for ATP-binding site mutants. Bacteria transformed with wild-type LmRPH, vector, or mutants were cultured in solid LB medium complemented with 0, 10, 40, 80, or 320 μg/mL RIF.

of these residues was validated by an in vitro enzymatic activity changes over time; therefore we used the RD and HD for the assay. The results show that the activity of Q183A is slightly detection of RIF binding.). lower than that of the wild-type LmRPH, the activities of T136A, The RIF-binding pocket is comprised mainly of hydrophobic Q309A, and R311A mutants are significantly reduced, and those residues. Residues Val333, Met359, and Val368 constitute a of other mutants (K22A, R117A, and E297A) are extremely low hydrophobic patch and contact the naphthol ring of RIF through (Fig. 3E). Accordingly, the RIF-resistance levels of these mu- van der Waals forces; residues Ile331, Ile370, Ile394, Met383, tants are reduced to different extents, except for the Q183A Leu387, Met823, Met491, Met488, Leu478, and Met673 stabilize mutant, in which resistance is comparable to that in wild-type the ansa chain of RIF through hydrophobic interactions (Fig. 4 F LmRPH (Fig. 3F). and G). Mutations V333A, V368A, M383A, or M673A increase the size of the pocket and reduce the phosphorylation activity of Both the RD and HD Are Involved in Rif Binding. The structure of the LmRPH, whereas V333W or V368W causes steric conflict and RD can be divided further into three subdomains: subdomain I almost abolishes the activity (Fig. 4H). The R group of RIF (α12–16, 28–30, and β14–18), II (α17–20 and 26–27), and III points toward the opening of the pocket and packs against res- (α21–25) (Fig. 4A). Searches of the Protein Data Bank failed to idues Pro356 and Phe479; replacement of either of these two identify any entry that is structurally homologous to the RD, residues with alanine has only minor effects on the phosphory- suggesting that the RD represents a previously unidentified lation activity and RIF binding (Fig. 4F and Table S1). This structural fold related to RIF binding. In the LmRPH–ANP–RIF finding likely explains why RPH can phosphorylate various structure, the HD binds with all three subdomains of the RD members of the rifamycin family that differ primarily at the R from the concave side and forms the RIF-binding pocket group (13). with subdomains I and II of RD. Distinct from the AD ATP- binding cleft, which faces the concave side of LmRPH, the Phosphorylation of RIF. The LmRPH–ANP–RIF complex struc- opening of the RIF-binding pocket faces the convex side (Figs. ture allows us to examine the phosphorylation site of RIF. The 2A and 4A). Structural comparison of LmRPH–ANP–RIF with previously identified phosphorylation site of RIF, C21 hydroxyl, LmRPHG527Y-ANP reveals significant conformational changes is about 6.7 Å away from residue His825 of the HD (Fig. 5A). A at the RIF-binding pocket (Fig. 4B). Specifically, the binding of water molecule between His825 and C21 hydroxyl forms a hy- the HD with the RD pushes away α14–α16 and connecting loops drogen bond with the C21 hydroxyl. When we modeled the of the RD, leading to a rearrangement of the surrounding in- product RIF-P into the structure (Fig. 5B), we found that the ward-facing residues that create an RIF-binding pocket (Fig. 4 phosphate group of RIF-P could form four hydrogen bonds with B–D). These structural observations suggest that both the RD Lys670, Arg666, and Gln337 and that the distance between RIF-P and HD are involved in RIF binding. Consistently, we found that and residue His825 is about 4 Å. These structural observations the RD alone is not sufficient to bind with RIF, but the RD and suggest that the HD residue His825 and residues Lys670, Arg666, HD together can bind RIF with high affinity (Kd = 79.4 μM) and Gln337 are involved in RIF phosphorylation. To verify this (Fig. 4E). (The binding affinity of RIF with full-length LmRPH possibility, we mutated these four residues to alanine and

3806 | www.pnas.org/cgi/doi/10.1073/pnas.1523614113 Qi et al. Downloaded by guest on September 28, 2021 Fig. 4. The RIF-binding pocket. (A) Structure of the RD (in LmRPH–ANP–RIF). The gray dashed lines separate three subdomains (I, II, and III) of the RD. RIF is shown as magenta sticks. (B) Conformational changes of the RD induced by HD binding. The RD (light blue) and HD (orange) of the LmRPH–ANP–RIF structure was superposed with that of the LmRPHG527Y–apo structure (gray). α14–16, which undergo conformational changes after HD binding, are highlighted in red. (C) Surface view of the RIF-binding pocket in the LmRPHG527Y–apo structure. (D) Surface view of the RIF-binding pocket in the LmRPH–ANP–RIF structure. The RD, HD, and α14–16 are colored light blue, orange, and red, respectively. (E) The RIF-binding affinity of the RD and RD–HD measured by ITC. Binding isotherms for RD and RD–HD are colored green and red, respectively. (F) Residues at the RIF-binding site. RIF (magenta) and interacting residues (light blue) are shown as sticks. (G) Chemical structure of RIF. The naphthol ring and ansa chain of RIF are shown in pink and blue, respectively, and the R group is outlined by a dashed box. (H) In vitro catalytic activity of RIF-binding mutants detected by HPLC. BIOCHEMISTRY determined their activities. The results show that the H825A, addition, positively charged residues often function as catalytic R666A, and K670A mutants lose the ability to phosphorylate bases to abstract a proton at the reaction centers of phosphate- RIF both in vitro and in vivo, and Q337A has significantly re- transfer and other enzymes, including MAPK, phos- duced activity (Fig. 5 C and D). Notably, residue His825 is highly phothreonine , IMP dehydrogenase, pectate/pectin , conserved among RPHs, reminiscent of the catalytic His residue fumarate reductase, and L-aspartate oxidase (21–23), suggesting in PEP synthase (20). Using the Phos-tag SDS/PAGE experiment, that Lys670 and Arg666 might be candidate residues for the cat- we found that LmRPH is phosphorylated in the presence of ATP alytic bases of LmRPH. but the H825A mutant is not (Fig. 5E). The phosphorylation of residue His825 is confirmed in an MS analysis (Fig. S6). These Discussion results suggest that this conserved His residue also may function as In this work we captured two major conformational states of the a phosphate acceptor and donor in the phosphorylation of RIF. In LmRPH catalytic process: a conformation in which the HD binds

Fig. 5. Catalytic center of RIF phosphorylation. (A) Catalytic site of RIF phosphorylation. Residues His825, Lys670, Arg666, and Gln337 and RIF are shown as sticks; the water molecule is shown as a red sphere. Distances between the water molecule and surrounding residues are shown as dashed lines. (B) Catalytic site with a modeled RIF-P. Distances between the phosphate group and residues are shown as dashed lines. (C) In vitro catalytic activity of H825A, K670A, R666A, and Q337A detected by HPLC. (D) E. coli growth assay of the mutants in C. Bacteria transformed with wild-type LmRPH, vector, or mutants were cultured in solid LB medium complemented with 0 or 10 μg/mL RIF. (E) Phosphorylation analysis of wild-type LmRPH and the H825A mutant using Phos-tag SDS PAGE. LmRPH-P, phosphorylated LmRPH.

Qi et al. PNAS | April 5, 2016 | vol. 113 | no. 14 | 3807 Downloaded by guest on September 28, 2021 the pyruvate-binding domain to transfer phosphate from ATP to pyruvate (Fig. S7) (24–26). Based on the structure of RIF bound to the RNAP core enzyme, the C21 hydroxyl of RIF points toward the inside of the RIF-binding pocket and forms hydrogen-bonding in- teractions with nearby residues (1). Phosphorylation of this hydroxyl may lead to steric clash, thereby weakening or abolishing the binding of RIF to RNAP and ultimately resulting in resistance to RIF. RPHs are widespread among Bacillales, Actinomycetales, and Clostridiales, which include many human pathogens. Searches of the pathogenic bacterial genomes of Bacillus anthracis, En- terococcus faecalis, Nocardia brasiliensis, and Listeria mono- cytogenes all reveal RPH , which may limit the clinical use of rifamycins against these . Our mechanistic study of LmRPH provides feasible strategies, such as developing high- affinity RPH inhibitors or new RIF derivatives that are not susceptible to RPH, to overcome RPH-mediated resistance. Materials and Methods See SI Materials and Methods for details. In general, LmRPH protein was expressed in E. coli and purified to homogeneity for crystallization. All data Fig. 6. Catalytic process of LmRPH-mediated RIF phosphorylation. were collected and processed with HKL3000 (27). The structures were de- termined using programs in Phenix (28), and structural models were built with Coot (29). The products of the enzymatic assay were detected with to the AD, and a conformation in which the HD binds to the HPLC/MS. The substrate-binding affinity was measured with ITC. Data col- RD. Structural-based analysis confirmed that the HD functions lection and refinement statistics are summarized in Table S2. as a toggle switch, swinging between the two distant domains. ACKNOWLEDGMENTS. We thank the staff members at the BL19U beamline When binding to the AD, the HD facilitates ATP binding and of the National Center for Protein Science Shanghai and the BL17U beamline hydrolysis, grabbing a phosphate by residue His825. Then the of the Shanghai Synchrotron Radiation Facility for technical assistance in HD swings over to the RD, facilitating RIF binding and initiating data collection and the staff at the core facility center of the Institute of Plant Physiology and Ecology for MS experiments and analysis. This work RIF phosphorylation. The dynamic nature of the LmRPH protein was supported by National Natural Science Foundation of China Grant enables the smooth transition between these two conformational 31322016 and National Program on Key Basic Research Projects Grant states (Fig. 6). This mechanism resembles that of three-domain 2015CB910900 and by funding from the National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, pyruvate orthophosphate dikinase (PPDK) enzymes in which the Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological His domain swivels between the nucleotide-binding domain and Sciences, CAS.

1. Campbell EA, et al. (2001) Structural mechanism for inhibition of bacterial 16. Hoshino Y, et al. (2010) Monooxygenation of rifampicin catalyzed by the rox gene polymerase. 104(6):901–912. product of Nocardia farcinica: Structure elucidation, gene identification and role in 2. Sensi P, Margalith P, Timbal MT (1959) Rifomycin, a new antibiotic; preliminary re- drug resistance. J Antibiot (Tokyo) 63(1):23–28. port. Farmaco, Sci 14(2):146–147. 17. Fischbach MA, Walsh CT (2009) Antibiotics for emerging pathogens. Science 3. Aristoff PA, Garcia GA, Kirchhoff PD, Showalter HD (2010) Rifamycins–obstacles and 325(5944):1089–1093. opportunities. Tuberculosis (Edinb) 90(2):94–118. 18. Wright GD (2007) The antibiotic resistome: The nexus of chemical and genetic di- 4. Sensi P (1983) History of the development of rifampin. Rev Infect Dis 5(Suppl 3): versity. Nat Rev Microbiol 5(3):175–186. S402–S406. 19. Ramaswamy S, Musser JM (1998) Molecular genetic basis of antimicrobial agent 5. Getahun H, Matteelli A, Chaisson RE, Raviglione M (2015) Latent Mycobacterium resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 79(1): – tuberculosis infection. N Engl J Med 372(22):2127 2135. 3–29. 6. Rothstein DM, van Duzer J, Sternlicht A, Gilman SC (2007) Rifalazil and other ben- 20. Narindrasorasak S, Bridger WA (1977) Phosphoenolypyruvate synthetase of Escher- zoxazinorifamycins in the treatment of chlamydia-based persistent infections. Arch ichia coli: Molecular weight, subunit composition, and identification of phosphohis- Pharm (Weinheim) 340(10):517–529. tidine in phosphoenzyme intermediate. J Biol Chem 252(10):3121–3127. 7. Huang DB, DuPont HL (2005) Rifaximin–a novel antimicrobial for enteric infections. 21. Li J, et al. (2014) Palladium-triggered deprotection chemistry for protein activation in J Infect 50(2):97–106. living cells. Nat Chem 6(4):352–361. 8. Schoenfeld P, et al. (2014) Safety and tolerability of rifaximin for the treatment of 22. Zhu Y, et al. (2007) Structural insights into the enzymatic mechanism of the patho- irritable bowel syndrome without constipation: A pooled analysis of randomised, genic MAPK phosphothreonine lyase. Mol Cell 28(5):899–913. double-blind, placebo-controlled trials. Aliment Pharmacol Ther 39(10):1161–1168. 23. Guillén Schlippe YV, Hedstrom L (2005) A twisted base? The role of arginine in 9. Dorman SE, Chaisson RE (2007) From magic bullets back to the magic mountain: The enzyme-catalyzed proton abstractions. Arch Biochem Biophys 433(1):266–278. rise of extensively drug-resistant tuberculosis. Nat Med 13(3):295–298. 24. Herzberg O, et al. (1996) Swiveling-domain mechanism for enzymatic phosphotransfer 10. Goldstein BP (2014) Resistance to rifampicin: A review. J Antibiot (Tokyo) 67(9): – 625–630. between remote reaction sites. Proc Natl Acad Sci USA 93(7):2652 2657. 11. Baysarowich J, et al. (2008) Rifamycin antibiotic resistance by ADP-ribosylation: 25. Nakanishi T, Nakatsu T, Matsuoka M, Sakata K, Kato H (2005) Crystal structures of Structure and diversity of Arr. Proc Natl Acad Sci USA 105(12):4886–4891. pyruvate phosphate dikinase from maize revealed an alternative conformation in the – 12. Spanogiannopoulos P, Thaker M, Koteva K, Waglechner N, Wright GD (2012) Char- swiveling-domain motion. Biochemistry 44(4):1136 1144. acterization of a rifampin-inactivating glycosyltransferase from a screen of environ- 26. Lim K, et al. (2007) Swiveling domain mechanism in pyruvate phosphate dikinase. – mental actinomycetes. Antimicrob Agents Chemother 56(10):5061–5069. Biochemistry 46(51):14845 14853. 13. Spanogiannopoulos P, Waglechner N, Koteva K, Wright GD (2014) A rifamycin in- 27. Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) HKL-3000: The in- activating phosphotransferase family shared by environmental and pathogenic bac- tegration of data reduction and structure solution–from diffraction images to an teria. Proc Natl Acad Sci USA 111(19):7102–7107. initial model in minutes. Acta Crystallogr D Biol Crystallogr 62(Pt 8):859–866. 14. Tupin A, et al. (2010) Resistance to rifampicin: At the crossroads between ecological, 28. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- genomic and medical concerns. Int J Antimicrob Agents 35(6):519–523. molecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221. 15. Wright GD (2005) Bacterial resistance to antibiotics: Enzymatic degradation and 29. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. modification. Adv Drug Deliv Rev 57(10):1451–1470. Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501.

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