Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

Abstract

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 Å resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC₅₀ ∼ 18 μM in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU, also referred to as UDP-GlcNAc pyrophosphorylase) is an essential, bifunctional enzyme that catalyzes the production of UDP-N-acetylglucosamine (UDP-GlcNAc), an important precursor in bacterial peptidoglycan and lipopolysaccharide biosynthesis (Anderson and Raetz 1987). In the first step of the reaction, GlmU catalyzes the transfer of the acetyl group from acetyl-CoA to glucosamine-1-phosphate (GlcN-1-P) to produce N-acetylglucosamine-1-phosphate (GlcNAc-1-P):

In the second reaction, uridyl monophosphate is transferred from UTP to GlcNAc-1-P to produce UDP-GlcNAc and pyrophosphate (PPi) (Mengin-Lecreulx and van Heijenoort 1993, 1994; Gehring et al. 1996; Pompeo et al. 1998):

GlmU owes its bifunctional role to two functionally autonomous active sites: the acetyltransferase active site and the uridyltransferase active site (Mengin-Lecreulx and van Heijenoort 1993, 1994; Gehring et al. 1996; Pompeo et al. 1998). Kinetic and structural studies demonstrated that the two active sites reside on two distinct protein domains. The acetyltransferase reaction is carried out within the C-terminal (acetyltransferase) domain, while the rate-limiting uridyltransferase reaction occurs within the N-terminal (uridyltransferase) domain. It has been previously shown that the uridyltransferase activity follows a sequential substrate-binding order with UTP binding first noncovalently to the GlmU enzyme (Mochalkin et al. 2007). Upon UTP binding, the uridyltransferase active site remains in an open apo conformation until GlcNAc-1-P binds and induces a large conformational change at the GlcNAc-binding site. Following the binding of GlcNAc-1-P to the UTP charged active site, the phosphate oxygen of GlcNAc-1-P performs a nucleophilic attack on the α-phosphate group of UTP to complete the reaction.

A series of crystal structures of GlmU determined in apo form and in complexes with the natural substrates as well as the product (Brown et al. 1999; Kostrewa et al. 2001; Olsen and Roderick 2001; Sulzenbacher et al. 2001; Mochalkin et al. 2007) laid the groundwork for characterization of the uridyltransferase active site. This site can be arbitrarily divided into two substrate-binding regions: (1) the uridine-binding region of UTP and (2) the GlcNAc-binding site of GlcNAc-1P. The uridine binding region consists of the A12–G14, R18, K25, Q76–T82, and D105 structural segments that form the uracyl and ribose binding subsites containing the key residues in UTP substrate recognition and catalysis. The GlcNAc-binding site is comprised of T82, T137–G140, N169–G171, and E195–T199 structural segments arranged to recognize the N-acetylglucosamine moiety and orchestrate structural rearrangements required to complete the uridyltransferase reaction.

Herein we present a 1.9 Å resolution crystal structure of the first, to our knowledge, synthetic small-molecule inhibitor of GlmU (see Scheme 1). Inhibitor 1 (IC₅₀ ∼ 18 μM in a racemic mixture) was identified through a high-throughput screen of hiGlmU which was configured to detect inhibitors of the uridyltransferase reaction. The molecule contains a central 3-aminopiperidine core (A-ring) substituted by the phenethyl group (B-ring), directly attached to the nitrogen atom of the piperidine ring, 4-chloro-phenyl (C-ring), and the methoxy-propyl side chain.

Scheme 1.

The determined crystal structure indicated that 1 occupied an allosteric site of the enzyme that includes a lipophilic pocket adjacent to the GlcNAc-1P binding region. Based on the information obtained from the crystal structure and the mechanistic model of the uridyltransferase reaction, we propose a mechanism of allosteric inhibition by 1 that provides a basis for structure-guided design of a new class of mechanism-based small-molecule inhibitors of GlmU.

Results and Discussion

Overall structure of hiGlmU complexes

The overall structure of hiGlmU complexed with the inhibitor 1 is similar to that of apo hiGlmU (PDB code 2v0h) previously reported (Mochalkin et al. 2007). The RMSD of all the superimposed Cα atoms (450 atom pairs) is 0.24 Å. Electron density is well defined for all residues of the bifunctional enzyme except M1–K3 and K454–K456 at the N- and C-terminal ends, which also are disordered in the other reported hiGlmU structures (Mochalkin et al. 2007). The final structure has excellent geometry as defined by PROCHECK (Laskowski et al. 1993). The Ramachandran plot showed that 365 (93.1%) residues of the total 392 nonglycine and nonproline residues occupied most favorable regions, 26 (6.6%) residues were in additionally allowed areas, and one residue, D146, was located in the generously allowed region. There were no residues in the disallowed area. Two octahedrally coordinated magnesium ions were detected on the crystallographic threefold axis in electron density maps at the locations corresponding to the magnesium and cobalt ions reported in the crystal structure of EcGlmU with UDP-GlcNAc and Acetyl-CoA (Olsen et al. 2007). The ions were coordinated by three water molecules and three D406-OD atoms of the hiGlmU homotrimer. The refined isotropic temperature factors of these magnesium ions were 19.9 Ų and 21.5 Ų (occupancy was set to 1.0).

Inhibitor binding mode

Inhibitor 1 binds to an allosteric site of hiGlmU that consists of a lipophilic pocket adjacent to the GlcNAc-binding site (Fig. 1A). The second part of the binding site includes a distal surface depression formed by an Asx-turn-ir motif containing residue N228 at the N-terminal end of the connecting α-helix. In hiGlmU, the lipophilic pocket and the surface depression area are separated by the solvent-exposed residue E224, which is a central point of the allosteric site. Oxygen atoms of E224 utilize hydrogen-bonded ionic interactions (2.7 Å) with the protonated nitrogen atom of the piperidinium (A-ring of the inhibitor, which is wedged between residues E224 and Y139 (Fig. 1B,C). The piperidinium ring makes interplanar angles of 90° and 165° with the 4-chloro-phenyl (C-ring) and the phenethyl group (B-ring) that occupy the distal surface depression and the lipophilic pocket, respectively. In addition to the hydrogen-bonded interactions with E224, the inhibitor is involved in another direct hydrogen-bonding contact with the terminal nitrogen atom of Q231 (3.3 Å). The OMIT electron density map of 1 is continuous and well defined for all atoms except for the methoxy-propyl side chain, which is pointed into the solvent area and disordered (Fig. 1B). In the lipophilic pocket, the benzyl ring of the phenethyl group is involved in the stacking interactions with Y139 (closest contact 3.7 Å) and hydrophobic contacts with the side chain of T170 (>4.0 Å, Fig. 1C). In addition, positively charged hydrogen atoms of the B-ring system are pointed toward the carbonyl oxygen atoms of residues N169 (3.6 Å) and V223 (3.4 Å). In the distal surface depression, the 4-chloro-phenyl C-ring forms hydrophobic contacts with the side chains of E224 (>4.0 Å), Q231 (>3.5 Å). Residue M221 and L235 are located at the bottom of the surface depression facing the chlorine atom of the C-ring (>3.0 Å).

Figure 1.

hiGlmU in complex with allosteric inhibitor 1. (A) View of the electrostatic surface potential of hiGlmU at the uridyltransferase active site and the allosteric region. Positive electrostatic potential is colored blue. Negative potential is colored red. Inhibitor 1 bound at the allosteric site is colored in the following atom colors: carbon, green; nitrogen, blue; oxygen, red; chlorine, orange. UDP-GlcNAc (PDB code 2v0i) shown at the substrate-binding site is colored in the following atom colors: carbon, yellow; nitrogen, blue; oxygen, red. (B) View of an OMIT electron density map contoured at 3σ level. (C) A schematic representation of inhibitor 1/hiGlmU interactions drawn using the MOE program (Chemical Computing Group, CCG). Hydrophobic residues are colored with a green interior; polar residues are colored in light purple. Basic residues are further annotated by a blue interior ring, and acidic residues with a red ring. The hydrogen-bonding interaction between the E224 side chain of hiGlmU and inhibitor 1 is drawn with a green arrowhead. (D) View of the superimposed hiGlmU coordinates from the complex with inhibitor 1 (colored in magenta), the product UDP-GlcNAc (colored in blue; PDB code 2v0i) and the apo form (colored in gold; PDB code 2v0h). In the UDP-GlcNAc and inhibitor 1 bound structures, the active site undergoes a substantial structural rearrangement, including the movement of residues N146, K156, and A160 by 5.5 Å, 5.9 Å, and 6.9 Å, respectively (distance measures between Cα pairs). Binding of inhibitor 1 prevents the structural rearrangement from open (apo-like) to closed (product-bound) conformations.

Proposed mechanism of allosteric inhibition of GlmU

A previously proposed mechanistic model of the uridyltransferase reaction of GlmU (Kostrewa et al. 2001; Mochalkin et al. 2007) along with the X-ray crystal structure of hiGlmU in complex with inhibitor 1 reported here provide insights into the mechanism of allosteric inhibition. To facilitate the enzymatic reaction, UTP binds first to the apo GlmU enzyme preserving the uridyltransferase active site in the open conformation. The crystal structure of hiGlmU complexed with UDP suggested (Mochalkin et al. 2007) that both β- and γ-phosphate groups of UTP are oriented into solvent. This allows the phosphate group of GlcNAc-1-P to attack the α-phosphate group of UTP that induces large conformational changes at the GlcNAc-binding subsite. The active site undergoes a substantial structural rearrangement when the entire region encompassing residues L133–G140 and V150–K166 tilts 20° to create a proper stereochemical environment for the formation of the product (Olsen and Roderick 2001; Kostrewa et al. 2001; Sulzenbacher et al. 2001). In the apo GlmU crystal structure (PDB code 2v0h), the distance between Y139-OH and E224-OE1 is 5.2 Å. As the uridyltransferase reaction progresses to the formation of the UDP-GlcNAc product, the lipophilic pocket is contracted as the result of structural rearrangements, and the distance between Y139-OH and E224-OE1 reduces to 2.9 Å (PDB code 2v0i). Because the inhibitor binds to the lipophilic pocket and interacts directly with the side chains of E224 and Y139, we propose that the mechanism of the allosteric inhibition of 1 results from the binding to the lipophilic pocket to obstruct conformational changes required for the phosphotransfer between substrates UTP and GlcNAc-1-P (Fig. 1D). As the crystal structure indicates, this binding of the central 3-aminopiperidine ring keeps the lipophilic pocket in an open conformation that is characterized by the distance between Y139-OH and E224-OE1 of 6.1 Å.

Implication for structure-based drug discovery and early SAR

The crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae determined at resolution 1.9 Å revealed an allosteric site that includes a lipophilic pocket adjacent to the GlcNAc-binding site. The inhibitory activity of 1 against hiGlmU suggests that the inhibitor prevents a conformational rearrangement at the uridyltransferase active site required for the catalytic reaction. As the protein–inhibitor interactions indicate, the inhibitor forms hydrogen-bonded ionic interactions between a protonated nitrogen atom of the piperidinium ring and the oxygen atoms of E224 in hiGlmU. Interestingly, 1 did not inhibit GlmU activity from the Gram-positive bacteria, such as Staphylococcus aureus or Streptococcus pneumoniae. GlmU from these organisms contains naturally occurring substitution of methionine or leucine for E224 in hiGlmU that leads to disruption of the key interaction with the protonated nitrogen atom of the piperidinium ring. These results provide a mechanistic insight into the bacterial specificity of the inhibitor and suggest that the allosteric site can be utilized for designing compounds with an improved selectivity profile among the GlmU orthologs.

A limited number of compounds were synthesized around inhibitor 1 to develop an early SAR (see Scheme 2). First, a substitution of 4-chloro to 4-fluoro of the ring C (compound 2) had almost no effect on the measured IC₅₀ (18 μM and 20 μM, respectively). Second, the lipophilic pocket was probed with a fluorine atom substituted in the ortho-, meta-, or para-positions of the phenyl ring B. The ortho- and meta-substitutions produced inactive compounds 3 and 4 (IC₅₀ > 100 μM), while the para-substitution led to a 4.5-fold decrease of potency (compound 5).

Scheme 2.

Modeling of these analogs in the crystal structure indicated that ortho-substitutions in ring B can result in close contacts with the side chain of L133 (∼2.5 Å) and E224 (∼2.7 Å); meta-substitutions can result in unfavorable contacts (∼2.5 Å) with the backbone carbonyl of V223 or N169, and the para-substitution can lead to a close (∼2.7 Å) contact with Cα of T170. Thus, none of the phenyl ring substitutions were well tolerated by the enzyme, emphasizing a lack of flexibility in the lipophilic region. Based on this early SAR, a number of valuable structure-based approaches can be suggested, including replacement of the phenyl ring B with a smaller ring. This would target potential hydrogen-bond interactions with the carbonyl oxygen of V223 and N169 within the lipophilic pocket and extend into the GlcNAc-binding region. Finally, the methoxy-propyl side chain of 1 can be shortened or removed, as it points out into the solvent region and is disordered in the crystal structure.

Materials and Methods

Synthesis of allosteric inhibitors of hiGlmU

The inhibitor 1 was identified through a high-throughput screen of hiGlmU configured to find inhibitors of the uridyltransferase reaction (see Scheme 3). Upon identification, 1 was resynthesized via standard chemistry starting from 3-keo-Boc-piperdine, which is outlined in Scheme 1. The final compound 1 was found to be a 18 μM inhibitor of hiGlmU as a racemic mixture. It was also assayed for binding with hiGlmU by biophysical techniques, such as isothermal denaturation and saturation transfer desorption (NMR) (data not shown).

Scheme 3.

Protein expression, purification, and crystallization

The higlmU gene was cloned by PCR from genomic DNA inserted into a pPW2 expression vector (Affinium Pharmaceuticals) and overexpressed in BL21 AI cells (Invitrogen) using a proprietary rich medium supplemented with 100 μg/mL ampicillin. Protocols used for protein expression and purification were previously described (Mochalkin et al. 2007). The purified protein was stored at −80°C in the buffer consisting of 10 mM HEPES (pH 7.5), 500 mM NaCl, and 0.2 mM TCEP. To prepare a protein–inhibitor complex, a stock solution of 100 mM compound was dissolved in dimethyl sulfoxide and mixed with the protein (14 mg/mL) to a final inhibitor concentration of 2.5 mM along with 20 mM MgCl₂. The complex was incubated at 4°C for 4 h before crystallization. The crystals were grown at ambient temperature using the hanging-drop vapor diffusion method. Four microliter drops consisted of equal parts of the protein solution containing inhibitor and the reservoir solution composed of 1.7–1.8 M ammonium sulfate, 2% PEG-400, and 0.1M MES (pH 5.4–6.1). The GlmU crystals were visible after 5 d and grew typically to a dimension of (0.20 × 0.15 × 0.07) mm.

X-ray data collection and structure determination

X-ray diffraction data were measured at the Advanced Photon Source facility on beamline 17-ID operated by the Industrial Macromolecular Crystallography Association. The crystal was mounted in a cryo-loop and treated with the cryo-protection solution as previously described (Mochalkin et al. 2007). Auto-indexing and processing of the measured intensity data were carried out with the HKL2000 software package (Otwinoski and Minor 1997). The intensity data collection statistics are summarized in Table 1. The crystal structure was determined using protein coordinates of the apo hiGlmU as the starting model (PDB ID 2v0h). The coordinates were optimized by rigid-body, coordinates, and B-value minimization using CNX 2002 (Brünger et al. 1998) and REFMAC 5.1 (Collaborative Computational Project, Number 4 1994). Calculated (2Fo-Fc) and (Fo-Fc) electron density maps were utilized for interactive fitting of protein structures into electron density using X-BUILD (Oldfield 2001a) implemented in QUANTA (Accelrys Inc). Placement of the ligands into electron density maps was carried out with X-LIGAND (Oldfield 2001b). Solvent water molecules were added periodically based on examination of difference density maps. Final coordinates were validated using PROCHECK (Laskowski et al. 1993). Summary of final refinement parameters and the final R _work and R _free values are listed in Table 1.

Table 1.

X-ray intensity data statistics, refinement summary of deviations from ideality, and the final R-values of hiGlmU in complex with 1

Inhibitor inhibition assay

Inhibition of enzyme activity was measured using a modified format of the MESG/PNPase coupled assay format for the continuous detection of the product inorganic phosphate described by Gehring et al. (1996). Using a 50-μL volume in a 384-well format, the standard assay mix contained the following: 40 mM HEPES (pH 7.5), 2.5 mM MgCl₂, 0.3 mM TCEP, 20 μM UTP, 20 μM GlcNAc-1-P, 10 μg/mL SipA, 0.05 U/mL Pnpase, 20 mM MESG, 1 mM citric acid, and 3 nM GlmU enzyme. The reaction was monitored for 5 min at A360 on a Spectromax plate reader.

Data deposition

Atomic coordinates have been deposited in the RCSB Protein Data Bank (accession code: 2vd4).