research papers

Acta Cryst. (2017). D73, 349–364 https://doi.org/10.1107/S2059798317002534 349

Received 9 November 2016

Accepted 14 February 2017

Edited by J. L. Martin, Griffith University,

Australia

Keywords: 8-oxoguanine nucleotides;

sanitization of nucleotide pool; Nudix enzyme;

histidine phosphatase domain; binding sites at

intermolecular interfaces; enzyme action;

MutT1; Mycobacterium smegmatis.

PDB references: Mycobacterium smegmatis

MutT1, 5gg5; complex with 8-oxo-dGTP, 5gg6;

complex with 8-oxo-dGTP, 8-oxo-dGMP and

pyrophosphate (I), 5gg7; complex with

8-oxo-dGTP, 8-oxo-dGMP and pyrophosphate

(II), 5gg8; complex with 8-oxo-GTP, 8-oxo-GMP

and pyrophosphate, 5gg9; complex with

8-oxo-GDP, 8-oxo-GMP and pyrophosphate,

5gga; complex with 8-oxo-dGDP, 5ggb;

complex with phosphate and magnesium ions

(excess magnesium) (I), 5ggc; complex with

phosphate and magnesium ions (excess

magnesium) (II), 5ggd

Supporting information: this article has

supporting information at journals.iucr.org/d

Biochemical and structural studies ofMycobacterium smegmatis MutT1, a sanitizationenzyme with unusual modes of association

S. M. Arif,a A. G. Patil,b U. Varshneyb and M. Vijayana*

aMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India, and bDepartment of Microbiology and

Cell Biology, Indian Institute of Science, Bangalore 560 012, India. *Correspondence e-mail: mv@mbu.iisc.ernet.in

Mycobacterium smegmatis MutT1, which is made up of a Nudix domain (domain

1) and a histidine phosphatase domain (domain 2), efficiently hydrolyses 8-oxo-

GTP and 8-oxo-dGTP to the corresponding nucleoside diphosphates and

phosphate in the presence of magnesium ions. Domain 1 alone hydrolyses

nucleoside triphosphates less efficiently. Under high concentrations and over

long periods, the full-length enzyme as well as domain 1 catalyses the hydrolysis

of the nucleoside triphosphates to the respective nucleoside monophosphates

and pyrophosphate. The role of domain 2 appears to be limited to speeding up

the reaction. Crystal structures of the apoenzyme and those of ligand-bound

enzyme prepared in the presence of 8-oxo-GTP or 8-oxo-dGTP and different

concentrations of magnesium were determined. In all of the structures except

one, the molecules arrange themselves in a head-to-tail fashion in which domain

1 is brought into contact with domain 2 (trans domain 2) of a neighbouring

molecule. The binding site for NTP (site A) is almost exclusively made up of

residues from domain 1, while those for NDP (site B) and NMP (site C) are at

the interface between domain 1 and trans domain 2 in an unusual instance of

intermolecular interactions leading to binding sites. Protein–ligand interactions

at site A lead to a proposal for the mechanism of hydrolysis of NTP to NDP and

phosphate. A small modification in site A in the crystal which does not exhibit

the head-to-tail arrangement appears to facilitate the production of NMP and

pyrophosphate from NTP. The two arrangements could be in dynamic

equilibrium in the cellular milieu.

1. Introduction

Reactive oxygen species and reactive nitrogen intermediates

produced during cellular metabolism or present as environ-

mental pollutants generate oxidatively damaged nucleoside

triphosphates (NTPs) and deoxynucleoside triphosphates

(dNTPs) in the cell (Cooke et al., 2003; Szabo & Ohshima,

1997; Hayakawa et al., 1999). Excessive accumulation of these

nucleotides in the nucleotide pool impairs the fidelity of DNA

replication (Pursell et al., 2008) and RNA transcription

(Kamiya et al., 2009). Of the nucleic acid bases, guanine, which

is highly susceptible to oxidation, is converted to 8-oxo-7,8-

dihydroguanine (8-oxo-G). Post-incorporation, 8-oxo-G is a

major source of spontaneous mutation because of its ambig-

uous and almost equally efficient pairing with cytosine and

adenine during replication (Kuchino et al., 1987; Moriya et al.,

1991; Shibutani et al., 1991). To deal with such challenging

threats to genomic integrity, cells have developed an elaborate

8-oxo-G (GO) repair system consisting of MutM (Fpg), MutY

and MutT (Kurthkoti & Varshney, 2011; Michaels & Miller,

1992). 8-Oxo-G paired with cytosine is excised by MutM, while

MutY removes adenine or guanine paired with 8-oxo-G

during replication (Au et al., 1989; Jain et al., 2007; Kurthkoti et

ISSN 2059-7983

# 2017 International Union of Crystallography

al., 2010). The MutT protein, on the other hand, works in a

completely different way by hydrolysing 8-oxo-G-containing

nucleotides in the nucleotide pool, thereby minimizing their

direct incorporation into DNA (Michaels & Miller, 1992).

MutT has been reported to have a high specificity for 8-oxo-G-

containing nucleotides. It hydrolyses 8-oxo-dGTP to 8-oxo-

dGMP at a rate three times faster than it hydrolyses dGTP to

dGMP (Maki & Sekiguchi, 1992). Thus, MutT substantially

minimizes errors in DNA replication and RNA transcription

caused by 8-oxo-G-containing nucleotides by sanitizing them

selectively from the nucleotide pool (Ito et al., 2005; Maki &

Sekiguchi, 1992; Taddei et al., 1997). MutT homologue 1

(MTH1), another 8-oxo-dGTPase, also hydrolyses 8-oxo-

dGTP to 8-oxo-dGMP (Nakabeppu et al., 2006).

MutT proteins are Nudix hydrolases, a versatile superfamily

of Mg2+-requiring enzymes that are found in almost every

form of life, including viruses, bacteria, archaea and eukary-

otes, and catalyse the hydrolysis of nucleoside diphosphate

linked to other moieties X, hence the name ‘Nudix’ (Bessman

et al., 1996). These enzymes are recognized by the highly

conserved 23-residue Nudix signature sequence or Nudix

motif, GX5EX7REUXEEXGU, where U is Leu, Ile or Val and

X is any residue (Bessman et al., 1996).

The susceptibility to guanosine modification in myco-

bacteria is very high owing both to the high GC content of

their genome and to the presence of reactive oxygen species

and reactive nitrogen intermediates produced by macrophages

as a host immune response following an infection. To deal with

this situation, mycobacterial genomes encode all of the

proteins specific to the GO repair pathway (Cole et al., 1998).

Of the nine proteins with a Nudix motif predicted by bio-

informatics studies, four putative Nudix hydrolases annotated

as MutT homologues, namely MutT1, MutT2, MutT3 and

MutT4, correspond to Escherichia coli MutT (EcMutT; Cole et

al., 1998; Dos Vultos et al., 2006; Moreland et al., 2009). Gene-

knockout studies have shown that both MutT1 and MutT4

have an antimutator role in Mycobacterium smegmatis (Dos

Vultos et al., 2006), while MutT3 (RenU) is an important

component of the redox homeostatic system, RHOCS, that is

critical for the survival of mycobacteria inside macrophages

and the formation of biofilms (Wolff et al., 2015). Subse-

quently, the involvement of M. tuberculosis MutT1 (MtMutT1)

in decreasing A to C mutations in a MutT-deficient strain of

E. coli was reported, thus suggesting an inherent antimutator

role for the enzyme (Patil et al., 2013). Moreover, MtMutT1

has been suggested to play a crucial role in the survival of the

bacteria under oxidative stress by the virtue of its antimutator

role (Patil et al., 2013). MutT1 and MutT2 have been shown to

have an 8-oxoguanosine triphosphatase activity (Dos Vultos et

al., 2006; Patil et al., 2013; Sang & Varshney, 2013). Interest-

ingly, MtMutT1 has been shown to hydrolyse 8-oxo-GTP and

8-oxo-dGTP to the corresponding nucleoside diphosphates,

but not to nucleoside monophosphates, at normal substrate

and enzyme concentrations (Patil et al., 2013). To understand

the basis of this novel and unusual activity and substrate

specificity, it was desirable to carry out structural studies on

MtMutT1. However, on account of problems with the

expression of MtMutT1, M. smegmatis MutT1 (MsMutT1) was

chosen for structural studies. Our preliminary studies on

MsMutT1 suggested the presence of an N-terminal Nudix

hydrolase domain (MsMutT1-NTD) corresponding to the

single-domain EcMutT and a C-terminal histidine phospha-

tase domain (MsMutT1-CTD) in the protein (Arif et al., 2012).

The presence of a phosphatase domain in MsMutT1 in addi-

tion to a Nudix hydrolase domain of the type that constitutes

EcMutT and human MutT homologue 1 (HsMTH1) was

intriguing. We have now carried out detailed crystallographic

studies of MsMutT1 and its complexes, along with comple-

mentary biochemical studies, as part of a long-range program

on the structural biology of mycobacterial proteins, which

forms a component of a concerted national and international

effort (Arora et al., 2011; Murillo et al., 2007; Terwilliger et al.,

2003). The protein appears to be an enzyme in which the

binding sites are formed primarily by intermolecular inter-

actions of the type that bring the Nudix domain of one

molecule and the phosphatase domain of a neighbouring

molecule into close proximity. The enzyme is capable of

hydrolysing 8-oxo-GTP and 8-oxo-dGTP to the corresponding

nucleoside diphosphates and monophosphates, simulta-

neously, sequentially or both. A detailed examination of the

crystal structures leads to a proposal as to how this is achieved.

2. Materials and methods

2.1. Cloning, expression and purification of MsMutT1,MsMutT1-NTD and MsMutT1-CTD

PCR amplification of the gene MSMEG_2390 (UniProt

reference No. A0QUZ2) from the M. smegmatis mc2155

genome was carried out by Phusion DNA polymerase using

the primers MsMutT1Fp (50-GATCCATATGTCGAAGGA-

CACCGA-30) and MsMutT1Rp (50-GTTGAAGCTTTACT-

TCTCGTC-30) containing NdeI and HindIII sites, respectively

(indicated in bold), as described previously for cloning of the

M. tuberculosis MutT1 (MtMutT1) gene (Patil et al., 2013).

The PCR amplicon was digested with NdeI and HindIII and

ligated into the pTrcNdeHis vector to generate pTrcNdeHis-

MsMutT1. The sequence of the construct was confirmed by

DNA-sequence analysis and it was then used to generate

clones corresponding to MsMutT1-NTD and MsMutT1-CTD.

The generation of clones corresponding to MsMutT1-NTD

and MsMutT1-CTD involved the PCR insertion of a STOP

codon followed by an NdeI restriction site between the

nucleotide sequences corresponding to the two domains in the

pTrcNdeHis-MsMutT1 construct. Site-directed mutagenesis

of pTrcNdeHis-MsMutT1 involving these insertions was

performed by PCR using Phusion DNA polymerase and the

primers MsMutT1(del)Fp (50-TTCGTAAAACGACCGTA-

ACATATGGTCGACACCAAGACGGT-30) and MsMutT1

(del)Rp (50-ACCGTCTTGGTGTCGACCATATGTTACG-

GTCGTTTTACGAA-30) with insertion sequences corre-

sponding to a STOP codon and an NdeI restriction site

(indicated in bold), employing the QuikChange protocol for

site-directed mutagenesis (Agilent). The mutant plasmid thus

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350 Arif et al. � Mycobacterium smegmatis MutT1 Acta Cryst. (2017). D73, 349–364

obtained was confirmed by restriction digestion using NdeI

and represents pTrcNdeHis-MsMutT1-NTD, a clone corre-

sponding to the N-terminal domain of MsMutT1. The

pTrcNdeHis-MsMutT1-NTD clone was then treated with the

NdeI enzyme, thereby removing the nucleotide sequence

corresponding to the N-terminal domain. This was followed

by separation and ligation of the digested clone, resulting in

pTrcNdeHis-MsMutT1-CTD, a clone corresponding to the

C-terminal domain of MsMutT1. The sequences of both of the

constructs were confirmed by DNA sequencing. A peptide

stretch, MGSSHHHHHHSSGLVPRGSH, containing the

histidine tag was appended to the N-terminus of all three of

the protein sequences during the cloning procedure.

Proteins were purified by Ni–NTA affinity chromatography

followed by size-exclusion chromatography using a Superdex

200 HiLoad 16/600 column and a buffer consisting of 25 mM

Tris–HCl pH 7.5, 500 mM NaCl, 10% glycerol, 2 mM

�-mercaptoethanol in conditions that were essentially iden-

tical to those described previously for MtMutT1 (Patil et al.,

2013).

2.2. Activity assays for MsMutT1, MsMutT1-NTD andMsMutT1-CTD

All three proteins were dialyzed extensively against a buffer

consisting of 25 mM Tris–HCl pH 7.5, 50 mM NaCl, 2%(v/v)

glycerol, 2 mM �-mercaptoethanol before performing an

activity assay. Activity assays were performed in assay buffers

and conditions similar to those used previously for MtMutT1

and human Nudix type 5 (NUDT5) proteins (Patil et al., 2013;

Ishibashi et al., 2003) with some modifications. Reaction

mixtures (10 ml) consisting of 25 mM Tris–HCl pH 7.5, 8 mM

MgCl2, 50 mM NaCl, 5 mM DTT, 2% glycerol, various NTP or

dNTP substrates (�100–1000 mM) and enzymes were incu-

bated at 310 K. Reactions were terminated by adding 10 ml of

0.1% SDS and the mixtures were analysed by separating them

using a DNAPac column (DNAPac PA200 analytical, 4 �

250 mm), employing an integrated high-pressure liquid-

chromatography (HPLC) system (UltiMate 3000). An elution

system consisting of buffer I (25 mM Tris–HCl pH 9.0) and

buffer II (1 M lithium chloride) with a gradient of 0–50% was

used at an isocratic flow rate of 0.5 ml min�1 for 28 min.

Nucleotides were detected by UV-light absorbance. Peaks

corresponding to guanosine and 8-oxoguanosine nucleotides

were detected at a wavelength of 252 nm.

2.3. Crystallization

The protein solution was dialyzed against a buffer

consisting of 25 mM Tris–HCl pH 7.5, 500 mM NaCl, 10%

glycerol, 2 mM �-mercaptoethanol prior to crystallization.

Crystals of apo MsMutT1 were obtained after extensive

screening using screening kits from Hampton Research

employing the microbatch-under-oil method at 298 K (Arif et

al., 2012). The quality of the crystals was improved by incu-

bating them in a drop containing the precipitant supplemented

with 20% ethylene glycol. Similar crystallization trials were

used to generate crystals of MsMutT1 complexed with the

known substrates 8-oxo-GTP and 8-oxo-dGTP (obtained from

Jena Bioscience) by the co-crystallization method. A mixture

consisting of the protein solution at a concentration of

7 mg ml�1 and the substrates at a molar concentration ten

times that of the protein was incubated for 2 h at 277 K before

crystallization. The above mixture supplemented with 10 mM

MgCl2 was also incubated in a similar fashion for another

set of crystallization experiments. Drops containing equal

volumes of the precipitant solution and the protein–substrate

mixture were used for crystallization. Eight further crystals

of MsMutT1 were generated during these crystallization

attempts using various precipitant solutions. Details of these

crystals, along with that of apo MsMutT1, and the respective

precipitants involved are given in Table 1.

2.4. X-ray data collection, structure solution and refinement

Intensity data from two of the nine crystals (crystal 3 and

crystal 8) were collected using a MAR345 detector mounted

on a Bruker-AXS Microstar Ultra II Cu K� rotating-anode

X-ray generator. Data sets from the remaining seven crystals

were collected using a CCD detector on the synchrotron X-ray

beamline BM14 at the European Synchrotron Radiation

Facility, Grenoble, France. All of the data sets were collected

at a temperature of 100 K using ethylene glycol or glycerol as

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Acta Cryst. (2017). D73, 349–364 Arif et al. � Mycobacterium smegmatis MutT1 351

Table 1Basic data on the different crystals.

See text for details. The values in parentheses in column 3 are the concentrations of magnesium ions in the medium.

Crystal No. LigandPrecipitant†(Mg concentration) Form

Head-to-tailarrangement Site A Site B Site C

1 None 1 I Yes2 8-Oxo-dGTP 2 II No 8-Oxo-dGTP3 8-Oxo-dGTP 2 III Yes 8-Oxo-dGTP PPi 8-Oxo-dGMP4 8-Oxo-dGTP 2 IV Yes 8-Oxo-dGTP PPi 8-Oxo-dGMP5 8-Oxo-GTP 2 IV Yes 8-Oxo-GTP PPi 8-Oxo-GMP6 8-Oxo-GTP (MgCl2 soaking) 2 (10 mM, 2 min) IV Yes 8-Oxo-GDP PPi 8-Oxo-GMP7 8-Oxo-dGTP (MgCl2 co-crystallization) 3 (10 mM) V Yes 8-Oxo-dGDP8 8-Oxo-dGTP 4 (200 mM) VI Yes9 8-Oxo-GTP 4 (200 mM) VI Yes

† 1, 0.2 M ammonium sulfate, 0.1 M MES monohydrate pH 6.5, 30%(w/v) PEG monomethyl ether 5000, 0.01 M TCEP hydrochloride; 2, 0.2 M sodium acetate trihydrate, 0.1 M Tris–HClpH 8.5, 30%(w/v) PEG 4000; 3, 0.2 M sodium acetate, 0.1 M sodium acetate trihydrate pH 4.6, 30%(w/v) PEG 4000; 4, 0.2 M magnesium chloride hexahydrate, 0.1 M Tris–HCl pH 8.5,30%(w/v) PEG 4000.

cryoprotectant. The data were processed and merged using

iMosflm (Battye et al., 2011) and SCALA (Evans, 2006) from

the CCP4 program suite (Winn et al., 2011). Structure-factor

amplitudes were obtained from intensities using TRUNCATE

from the CCP4 program suite.

The crystal structure of apo MsMutT1 was determined by

molecular replacement with Phaser (McCoy et al., 2007) from

the CCP4 program suite using a data set collected previously

(Arif et al., 2012). A value of 2.32 A3 Da�1 for the Matthews

coefficient suggested the presence of one molecule in the

asymmetric unit (Matthews, 1968). The coordinates of puta-

tive MutT1 from Bifidobacterium adolescentis (PDB entry

3fjy; New York SGX Research Center for Structural Geno-

mics, unpublished work), with a sequence identity of 28% to

MsMutT1, were used as the search model to obtain the initial

phases. The solution obtained from Phaser was then built into

the partially interpretable electron-density map using Coot

(Emsley et al., 2010). An initial rigid-body refinement followed

by positional refinement and individual B-factor refinement

was performed on this partially built solution using

REFMAC5 (Murshudov et al., 2011). Cycles of model building

and subsequent refinement followed. The structure thus

obtained was further refined using the high-resolution X-ray

data obtained subsequently. The coordinates of this structure

were then used as a search model to determine the structures

of the various MsMutT1–ligand complexes from the remaining

crystals by the molecular-replacement method. The structures

underwent similar refinement as described previously. The

2Fo � Fc and Fo � Fc maps computed at stages where R and

Rfree had acceptable values showed unambiguous density for

the respective ligands. The ligand molecules were subse-

quently modelled and their locations were further confirmed

by computing simulated-annealing Fo � Fc OMIT maps using

CNS v.1.3 (Brunger et al., 1998). Various components from the

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352 Arif et al. � Mycobacterium smegmatis MutT1 Acta Cryst. (2017). D73, 349–364

Table 2Details of data collection and refinement.

Values in parentheses are for the highest resolution shell.

Crystal No. 1 2 3 4 5 6 7 8 9

Data collectionSpace group P212121 P21 P21 P21 P21 P21 P212121 P21 P21

Unit-cell parametersa (A) 37.45 57.11 42.00 42.05 42.01 41.73 42.49 38.99 38.95b (A) 87.66 89.50 85.35 84.81 83.95 84.30 87.89 87.67 87.60c (A) 101.28 59.93 87.43 43.52 44.00 43.58 95.20 90.68 90.53� (�) 90.0 99.1 94.1 93.6 93.0 94.0 90.0 99.9 100.1

Resolution range (A) 43.85–1.64(1.73–1.64)

49.36–1.75(1.84–1.75)

29.90–1.70(1.79–1.70)

43.43–1.85(1.95–1.85)

43.94–1.60(1.69–1.60)

43.47–1.75(1.84–1.75)

64.59–1.10(1.16–1.10)

31.97–1.85(1.95–1.85)

44.57–1.70(1.79–1.70)

VM (A3 Da�1) 2.18 1.98 2.05 2.03 2.03 2.0 2.3 2.0 2.0Solvent content (%) 43.5 37.9 40.0 39.4 39.4 38.6 47.2 38.5 38.3No. of molecules in

asymmetric unit1 2 2 1 1 1 1 2 2

Unique reflections 41476 (5726) 59643 (8608) 66623 (9503) 26082 (3818) 40162 (5870) 30343 (4381) 145101 (20940) 51169 (7409) 65728 (9517)Multiplicity 3.6 (3.2) 5.0 (5.0) 4.5 (4.4) 4.2 (4.2) 4.5 (4.5) 5.9 (5.8) 7.9 (7.0) 5.3 (5.2) 3.2 (3.1)Completeness (%) 99.4 (96.3) 99.4 (98.8) 98.5 (96.5) 100.0 (100.0) 99.9 (100.0) 100.0 (100.0) 100.0 (99.9) 99.8 (99.3) 99.9 (100.0)hI/�(I)i 11.4 (3.6) 9.8 (1.9) 7.9 (1.8) 9.5 (2.0) 10.8 (2.1) 9.1 (1.6) 10.2 (1.9) 7.6 (1.9) 6.6 (1.5)Rmerge† (%) 7.1 (33.7) 8.7 (67.5) 8.6 (78.0) 8.6 (68.3) 7.3 (65.3) 11.2 (98.0) 9.8 (96.2) 13.1 (79.8) 7.9 (68.0)CC1/2 0.996 (0.832) 0.996 (0.803) 0.998 (0.593) 0.993 (0.770) 0.996 (0.800) 0.993 (0.706) 0.996 (0.720) 0.997 (0.744) 0.996 (0.789)

Refinement and model statisticsR factor (%) 15.7 16.2 19.6 17.6 16.5 17.8 16.0 22.3 21.6Rfree (%) 19.7 20.4 23.6 22.0 20.6 22.9 18.5 26.7 25.9R.m.s. deviation from ideal

Bond lengths (A) 0.018 0.019 0.015 0.017 0.016 0.018 0.02 0.015 0.017Bond angles (�) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 1.7 1.9

No. of non-H atomsProtein atoms 2441 4686 4617 2334 2357 2352 2511 4598 4594Ligands (NTP/NDP/

NMP/PPi)— 64 178 65 67 63 28 — —

Solvent atoms 457 562 644 262 339 239 486 484 527Average B factors (A2)

Protein atoms 15.2 30.8 26.7 37.0 27.5 35.9 15.9 29.4 30.7Ligands (NTP/NDP/

NMP/PPi)— 27.7 38.3 46.1 28.8 45.7 17.5 — —

Solvent atoms 32.7 39.0 35.6 42.8 38.2 42.8 31.6 34.2 36.6Ramachandran plot statistics‡ (residues in, %)

Favoured region 91.5 90.9 91.7 91.6 90.7 91.1 91.9 89.8 91.4Allowed region 8.5 9.1 7.7 8.4 8.9 8.9 7.7 10.0 8.2Generously allowed

region0.0 0.0 0.6 0.0 0.4 0.0 0.4 0.2 0.4

Disallowed region 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0PDB code 5gg5 5gg6 5gg7 5gg8 5gg9 5gga 5ggb 5ggc 5ggd

† Rmerge =P

hkl

Pi jIiðhklÞ � hIðhklÞij=

Phkl

Pi IiðhklÞ, where Ii(hkl) is the ith observation of reflection hkl and hI(hkl)i is the weighted average intensity for all i observations of

reflection hkl. ‡ Calculated for nonglycine and nonproline residues using PROCHECK.

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Acta Cryst. (2017). D73, 349–364 Arif et al. � Mycobacterium smegmatis MutT1 353

Figure 1HPLC separation of the nucleotide substrates and the products formed by the action of the enzyme using a DNAPac column (DNAPac PA200 analytical,4 � 250 mm). (a) Activity assay using full-length MsMutT1. (i)–(iii) are controls for (iv), (v) for (vi), (vii) for (viii) and (ix) for (x). (b) Activity assayusing MsMutT1-NTD (i, ii) and MsMutT1-CTD (iii, iv). (c) Effect on catalysis of a higher concentration and longer incubation of full-length MsMutT1(ii) and MsMutT1-NTD (iii) with the substrates. (i) is the common control for (ii) and (iii). (d) Analysis of the crystallization solution used during co-crystallization of MsMutT1 with 8-oxo-GTP/8-oxo-dGTP. (i) is the control for (ii). The MgCl2 concentration was 8 mM in (a) and (b), while it was200 mM in (c). MgCl2 was not added in (d). In all of the figures, the x axis is the retention time (min) of the nucleotides and the y axis is intensity of thenucleotide peak in milli-absorbance units (mAU) at a wavelength (WVL) of 252 nm.

precipitant and the cryoprotectant were also modelled into the

electron density when appropriate. O atoms of water mole-

cules were successively added to the structures in the final

cycles of refinement using peaks with heights greater than 3.0�and 1.0� in Fo � Fc and 2Fo � Fc maps, respectively. Alter-

native conformations were assigned to the side chains of a few

of the amino-acid residues and one ligand molecule in one of

the crystals during the course of model building and refine-

ment. In the cases where the data set had a resolution better

than 1.5 A, anisotropic B factors were used in the final stage of

refinement. Refinement parameters, along with data-collec-

tion and data-processing statistics, are summarized in Table 2.

2.5. Analysis of the structures

The refined structures were evaluated using PROCHECK

(Laskowski et al., 1993). Secondary structure was assigned

using STRIDE (Heinig & Frishman, 2004). Interatomic

distances were calculated using

CONTACT from the CCP4

program suite. Hydrogen bonds

were assigned based on a distance

less than or equal to 3.6 A

between the donor (D) and the

acceptor (A) atom and a D–

H� � �A angle greater than 90�.

Structural superpositions were

performed using ALIGN (Cohen,

1997). The buried surface area

was calculated using NACCESS

(Hubbard & Thornton, 1996).

Figures for molecular repre-

sentations were generated using

PyMOL (DeLano, 2002).

Topology and parameter files for

various nucleotides were gener-

ated by ACPYPE (AnteChamber

PYthon Parser interfacE; Sousa

da Silva & Vranken, 2012).

Energy minimizations of the

protein–ligand complexes were

performed using the GROMACS

v.5.0.7 package employing the

AMBER99SB force field

(Hornak et al., 2006). Free ener-

gies of ligand binding to the

protein were estimated using

AutoDock 4.2 (Morris et al.,

2009).

2.6. Dynamic light-scatteringmeasurements

A dynamic light-scattering

(DLS) experiment on MsMutT1

at three different concentrations

including one corresponding to

that used for crystallization

experiments (7 mg ml�1) was carried out at the Centre for

Cellular and Molecular Biology (CCMB), Hyderabad using a

SpectroSize 300, a cuvette-based DLS instrument employing a

laser diode operating at a wavelength of 660 nm, a scattering

angle of 90� and an avalanche photodiode detector. The

protein solutions were centrifuged at 13 000 rev min�1 prior to

the DLS measurements. 20 measurements were made for each

sample during the DLS experiments. The collected data were

analysed using the SpectroSize 300 software, resulting in

average hydrodynamic size-distribution profiles. Deconvolu-

tion of the autocorrelation function was performed using the

CONTIN algorithm (Provencher, 1982).

2.7. Small-angle X-ray scattering (SAXS) measurements

SAXS measurements of MsMutT1 in a buffer as used for

crystallization were recorded, again at CCMB, using an

S3-MICRO Point-Focus system (Hecus X-ray Systems),

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354 Arif et al. � Mycobacterium smegmatis MutT1 Acta Cryst. (2017). D73, 349–364

Figure 2Overall structure of MsMutT1. (a) Domain architecture of MsMutT1 and structural features of (b)MsMutT1-NTD (domain 1) and (c) MsMutT1-CTD (domain 2). Domain 1 is in red and domain 2 is ingreen. The linker region is in blue. The highly conserved Nudix motif in MsMutT1-NTD, which adopts acharacteristic SLHL (strand–loop–helix–loop) structure, and the conserved RHG motif in MsMutT1-CTDare shown in yellow.

employing an X-ray wavelength of 1.5418 A and a PILATUS

100K detector with a pixel size of 172 � 172 mm at a distance

of 300 mm from the sample. Scatterings were measured with

an exposure time of 2 h. Averaging of the raw data was carried

out using Fit2D (Hammersley, 1997). The data were analysed

using the ATSAS (Konarev et al., 2006) program suite and

scattering plots were generated using PRIMUS (Konarev et al.,

2003) from the same suite.

3. Results and discussion

3.1. Enzyme activity

The enzymatic activity of MsMutT1 against GTP and dGTP

and their corresponding oxidized forms, namely 8-oxo-GTP

and 8-oxo-dGTP, were analysed in identical assay conditions

involving an assay buffer similar to that used previously for

MtMutT1 and human Nudix type 5 (NUDT5) proteins (Patil et

al., 2013; Ishibashi et al., 2003), consisting of 25 mM Tris–HCl

pH 7.5, 8 mM MgCl2, 50 mM NaCl, 5 mM DTT, 2% glycerol

and an enzyme concentration of 0.27 mM (0.01 mg ml�1).

MsMutT1 hydrolyses 8-oxo-GTP and 8-oxo-dGTP to 8-oxo-

GDP and 8-oxo-dGDP, respectively (Fig. 1a); hydrolysis of

GTP and dGTP was not observed (Fig. 1a). 8-Oxo-GDP or

8-oxo-dGDP did not hydrolyse any further even after incu-

bation with the enzyme for 12–24 h.

The presence of a C-terminal histidine phosphatase domain

(MsMutT1-CTD) in addition to the N-terminal Nudix

hydrolase domain in MsMutT1 (MsMutT1-NTD) necessitated

an examination of the role of the phosphatase domain in the

enzyme. Thus, the enzymatic activities of the two domains

individually against 8-oxo-GTP and 8-oxo-dGTP were also

checked. Surprisingly, MsMutT1-NTD could hydrolyse both

the substrates to the corresponding nucleoside diphosphates.

However, it showed a diminished activity compared with the

full-length enzyme (Fig. 1b). For instance, 2.7 pmol of

MsMutT1 took approximately 30 min to hydrolyse 1 nmol of

8-oxo-dGTP completely. The same amount of substrate,

however, required 21.7 pmol of MsMutT1-NTD (approxi-

mately eight times that of MsMutT) and almost 4 h, rather

than just the 30 min required by the full-length enzyme, for

complete hydrolysis. MsMutT1-CTD, on the other hand, did

not show any activity towards either 8-oxo-GTP or 8-oxo-

dGTP (Fig. 1b). In another experiment, a mixture of the two

domains instead of the full-length protein was used in the

assay. The result of the experiment was identical to that

obtained when MsMutT1-NTD alone was used (Supplemen-

tary Fig. S1). Thus, it appears that although it is inactive

against either of the substrates, the presence of the C-terminal

histidine phosphatase domain is necessary as an integral part

of the enzyme molecule for efficient catalysis by the N-

terminal Nudix hydrolase domain.

3.2. Effect of enzyme concentration and incubation period

The results of crystallographic studies (see below) necessi-

tated an examination of the effects of enzyme concentration

and the incubation period on activity. Enzyme activity was

previously measured, as is usually performed, at compara-

tively low protein concentration and short incubation periods.

Crystallization, however, involves high protein concentrations

and long incubation periods. Varying amounts (0–200 mM) of

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Figure 3Arrangement of molecules and generation of ligand-binding sites. (a) Head-to-tail arrangement of MsMutT1 molecules that brings domain 1 and transdomain 2 into periodic proximity, leading to the generation of ligand-binding sites, as observed in most of the crystals. (b) Disposition of neighbouringmolecules in crystal 2. (c) Various ligand-binding sites in domain 1 and at the interface between domain 1 and trans domain 2. Ligands from differentcrystals are mapped approximately on the protein structure involving domain 1 and trans domain 2 of crystal 5.

magnesium were added in the crystallization experiments.

Crystals grown from solutions containing no externally added

magnesium were of great use in defining the different binding

sites and they probably represented intermediate stages in the

reaction process. Therefore, it was important to explore the

reaction over long periods in solutions containing a high

protein concentration and no externally added magnesium as

well.

MsMutT1 at a concentration of approximately 22 mM

(0.8 mg ml�1) converted 8-oxo-dGTP completely to 8-oxo-

dGMP after incubation for approximately 12 h in the presence

of 200 mM MgCl2 (Fig. 1c). A similar result was obtained

when 8-oxo-GTP was used instead of 8-oxo-dGTP. When the

experiment involving 8-oxo-dGTP was repeated with an

incubation period of 3 h, 8-oxo-dGDP and 8-oxo-dGMP were

obtained, with the former as the major product. A similar

result was obtained when 42.56 mM (0.8 mg ml�1) MsMutT1-

NTD was incubated for 12 h with 8-oxo-dGTP (Fig. 1c). An

identical experiment involving MsMutT1-CTD did not yield

any product. It would thus appear that in the presence of

magnesium the enzyme converts 8-oxoguanine trinucleotides

to the corresponding dinucleotides at low protein concentra-

tions and short periods of incubation. With increasing

concentrations and times of incubation, the mononucleotides

also begin to appear. When the time of incubation is further

increased, the trinucleotide is converted completely into the

mononucleotide. Thus, it seems that the trinucleotide is

converted first to the dinucleotide, which is further converted

to the mononucleotide. However, a direct conversion of

trinucleotide to mononucleotide simultaneously cannot be

ruled out. Furthermore, MsMutT1-NTD, but not MsMutT1-

CTD, is by itself capable of converting an NTP into an NDP as

well as an NMP, although sluggishly.

Crystallization experiments were carried out at a still higher

concentration of the enzyme, in most cases with no externally

added magnesium. Trace amounts of magnesium probably

copurified with the protein during preparation. Therefore,

most of the crystals were grown in conditions in which reac-

tions could presumably take place very slowly or partially on

account of the very limited availability of magnesium. The

formation of 8-oxo-GMP/8-oxo-dGMP along with 8-oxo-

GDP/8-oxo-dGDP was observed when appropriate solutions

used for co-crystallization of MsMutT1 with 8-oxo-GTP/

8-oxo-dGTP were analysed by HPLC (Fig. 1d).

3.3. Molecular structure: overall features

The structures of apo MsMutT1 and eight of its complexes,

determined at resolutions ranging from 1.85 to 1.10 A,

belonged to six somewhat related crystal forms (Tables 1 and

2). Four of the crystals have two molecules with nearly iden-

tical structures in the asymmetric unit. Thus, the structures

provide 13 independent copies of the MsMutT1 molecule. No

electron density was observed for the 20–22 N-terminal resi-

dues in all of the structures. Residues 35–46 are also ill-defined

to different extents in different molecules. Thus, the refined

models of the molecule consist of residues 289–302 of the 322

residues in the sequence.

All 13 copies of the molecule have essentially the same

structure, with a N-terminal domain (NTD) extending to

residue 157 and a C-terminal domain (CTD; residues 163–322)

connected by a linker region (residues 158–162) (Fig. 2a). The

NTD, which is made up of three �-helices (�1–�3) and six

�-strands (�1–�6), has an �/�/� sandwich fold (Fig. 2b). The

three long strands (�1, �4 and �5) form a curled mixed

�-sheet, which is sandwiched between a short �-helix (�3) on

one side and a long �-helix (�1) on the other side. This

corresponds to the main frame of the Nudix fold. The three

short antiparallel �-strands �2, �3 and �6 together with the

helix �2 form an additional lobe attached to this frame.

Residues 66–88 involving a short strand (�30), a loop, �1 and

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356 Arif et al. � Mycobacterium smegmatis MutT1 Acta Cryst. (2017). D73, 349–364

Figure 4Aggregation of MsMutT1 in solution. Scattered intensity versushydrodynamic radius in DLS experiments at protein concentrations of(a) 1 mg ml�1 and (b) 7 mg ml�1 in the crystallization buffer. (c) Guinierplot of a SAXS experiment at a protein concentration of 18 mg ml�1 inthe crystallization buffer. A departure from linearity indicates aggrega-tion (Putnam et al., 2007).

another loop (SLHL) constitute the highly conserved

23-residue MutT signature sequence or Nudix motif. The

CTD, which is primarily made up of a six-stranded �-sheet

(�1–�6) flanked by two helices on one side (�2 and �3) and

another set of two helices (�5 and �6) on the other side, has an

�/� architecture (Fig. 2c) of the type found in the members of

the phosphoglycerate mutase family (histidine phosphatase

superfamily, branch 1). The structure contains two more short

helices, namely �1 and �4. The loop connecting �1 and �1

harbours the conserved RHG (arginine–histidine–glycine)

motif, which is a distinctive feature of the histidine phospha-

tase superfamily.

Not only the geometries of the two domains but also their

mutual orientation remains nearly the same in all of the

crystals, presumably on account of the strong interaction

between the two domains in the molecule. The interface

between the NTD and CTD involves the burial of 1201.7 A2 of

surface area, which is nearly equally distributed between the

two domains, of which 714.5 A2 is nonpolar. In addition, the

two domains are also connected by eight distinct hydrogen

bonds.

3.4. Intermolecular interactions and generation of bindingsites

Among the crystals studied, all except crystal 2 exhibit

nearly the same type of aggregation of molecules. The central,

common feature of this aggregation pattern is a head-to-tail

arrangement around a 21 screw axis, in which domain 1 of one

molecule is brought into close periodic proximity to domain 2

of a neighbouring molecule (trans domain 2; Fig. 3a). The

packing of molecules in crystal 2 is very different and involves

proximity of the same domains from neighbouring molecules

(Fig. 3b). Domain 1 and trans domain 2, as they occur in the

head-to-tail arrangement, probably form the predominant

functional unit of the enzyme. The results of dynamic light-

scattering experiments on MsMutT1 at a protein concentra-

tion of 1 mg ml�1 (Fig. 4a) indicate a dimer (radius 39 A and

molecular weight 75.3 kDa) as the predominant species. This

observation is compatible with the occurrence of dimeric units

in a head-to-tail arrangement. The experiments were repeated

at protein concentrations of 4 and 7 mg ml�1. Both experi-

ments lead to an indication of the same pattern of higher order

aggregation (Fig. 4b). The pattern involves two broad peaks.

The first peak, with an approximate radius of 76 A, roughly

corresponds to a decamer. The second broadest peak, with an

approximate radius of 1330 A, corresponds to aggregates

involving a few thousand molecules. The occurrence of two

peaks cannot be readily explained in terms of the structure.

However, the results of light-scattering experiments indicate

that the protein forms larger aggregates, presumably involving

dimers, at higher concentrations. This observation is in

consonance with the aggregation pattern observed in the

crystal structure. SAXS analysis

of the protein was also carried

out. Interpretable intensities

could be obtained only at a very

high concentration (18 mg ml�1)

of the protein. The results of the

SAXS analysis again indicate

aggregation (Fig. 4c).

In the head-to-tail arrange-

ment in crystals of the apoenzyme

(crystal 1), the interaction

between domain 1 and trans

domain 2 involves the burial of

1422 A2 of surface area, of which

706 A2 is hydrophobic. The

extent of burial is comparable to

the burial of surface area

observed in the association of

subunits in some multimeric

proteins (Arif & Vijayan, 2012;

Jones et al., 2000; Jones &

Thornton, 1996). The interaction

also encompasses 12 hydrogen

bonds involving the main-chain

and side-chain atoms of Arg57,

Tyr58, Asp59, Met140, Asp141,

Leu143, Gln144, Tyr145 and

Arg149 of domain 1 and those of

Arg169, Gly174, Arg175, Arg176,

Lys273, Glu280, Asn295, Arg296

and Lys297 of trans domain 2.

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Figure 5Simulated-annealing Fo� Fc OMIT maps of representative ligands observed at sites A, B and C in differentcrystals. The OMIT maps are contoured at the 3� level. The crystals in which a particular ligand is observedare given in parentheses.

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358 Arif et al. � Mycobacterium smegmatis MutT1 Acta Cryst. (2017). D73, 349–364

Except in the preparation of crystals of the apoenzyme,

8-oxo-GTP or 8-oxo-dGTP was added to the medium in

crystallization experiments, along with varying amounts (0 to

200 mM) of MgCl2. The distribution of the ligand or the

products at different binding sites (A, B and C) in the domain

1–trans domain 2 complex in the crystals is indicated in

Fig. 3(c) and elaborated in Fig. 5. In crystal 2, which does not

exhibit the head-to-tail arrangement, only site A exists, which

is occupied by the ligand along with what appears to be a

magnesium ion in the neighbourhood interacting with the

terminal phosphate group of the ligand and other relevant

residues (see later). In crystals 3, 4 and 5 site A is occupied by

the ligand and a magnesium ion in the neighbourhood, site B

by a pyrophosphate group (PPi) and site C by an appropriate

nucleoside monophosphate. Magnesium ions were not added

during the preparation of these crystals. The presence of what

could be a magnesium ion at site A might have been part of

the purified protein. Crystal 6 was obtained by soaking crystal

5 in a solution of 10 mM MgCl2 in the mother liquor for 2 min

and then cooling the crystal. The occupancies of sites B and C

are unaffected. However, site A is now occupied by 8-oxo-

GDP, indicating that a stage in the process of hydrolysis has

been captured. 10 mM MgCl2 was added to the solution from

which crystal 7 grew. In this crystal only site B is occupied, now

by 8-oxo-dGDP. Crystals 8 and 9 were grown in a medium

containing excess MgCl2. No nucleotide is observed at any site.

Instead, there is a cluster involving a phosphate group, three

magnesium ions and water molecules at a location close to site

A. The situation in these crystals could represent one in which

the reaction is complete. It may be mentioned that clusters

involving three magnesium ions and a phosphate at similar

locations have also been observed in other structures

containing the Nudix motif (Bailey et al., 2002; Messing et al.,

2009; Nakamura et al., 2010). Thus, while crystal 2 and crystals

8 and 9 could represent the beginning and the end of the

process, respectively, crystals 3, 4, 5, 6 and 7 appear to

represent intermediate situations that occur when the avail-

ability of magnesium ions is limited.

Site A is made up of amino-acid residues Arg55–Lys67,

Val99–Ile103, Lys108–Tyr112, Tyr145–Asp148, Glu81 and

Glu85, all of which belong to domain 1. A short stretch from

trans domain 2 involving Gly272, Lys273 and Pro276 also

occurs at one edge of the site in all relevant crystals except

crystal 2. The interactions of the protein with the nucleoside

triphosphate which occur in the relevant complexes are

illustrated in Fig. 6(a). Ligand binding involves the burial of

332–345 and 58–64 A2 of the surface areas of domain 1 and

Figure 6Protein–ligand interactions and substrate specificity. Interactions of protein with ligand at (a) site A, (b) site B and (c) site C. Dipoles of aromatic groupsare indicated in (a). In this and subsequent figures, residues from trans domain 2 are distinguished by asterisks.

trans domain 2, respectively. The dominant interactions are

between the triphosphate tail and the basic side chains of

Arg55, Arg57, Lys65, Lys67 and Lys108. The side-chain

hydroxyls of Tyr58 and Tyr101 also interact with the tail. O20

of the sugar interacts with the side chains of Asp60 and Tyr145

when the ligand is 8-oxo-GTP. Only Asp148 interacts with the

base through hydrogen bonds. The base is, however, sand-

wiched between the aromatic side chains of Tyr101 and

Tyr145. Residues from trans domain 2 only make water-

mediated interactions with the NTP or NDP ligand. These

water bridges do not exist in crystal 2, which does not exhibit

domain 1–trans domain 2 interactions. This difference in

molecular association does not affect the geometry of site A in

crystal 2, except for a slight shift in the location of the ligand.

However, in crystal 2 the 124–128 stretch, particularly Glu127,

moves close to the ligand. This movement appears to have

functional implications (see below).

The charge distribution in and hence the electrostatic

potential around the base appears to be important in discri-

minating between 8-oxo-G and G for binding at site A. The

orientation of dipoles, of the type involved in the case of

human OGG1 (David et al., 2007), could be central in this

discrimination. As illustrated in Fig. 6(a), the orientation of

the dipoles in 8-oxo-G and the flanking tyrosine side chains

are such as to promote strong interaction of the base with the

side chains. The interaction is weakened when the base is G, in

which the direction of the dipole is inclined with respect to

that in 8-oxo-G. The substitution of an O atom at C8 promotes

the protonation of N7, which facilitates the formation of a

water bridge with Asp147. Furthermore, the observed water

bridge between O8 and Pro102 O is possible only when 8-oxo-

G, instead of G, is the ligand.

The bulk of the residues which constitute site B are from

trans domain 2. In particular, residues 169–176, which form

part of a long loop, and the short loop 271–273 are involved in

interactions with PPi or the diphosphate tail of 8-oxo-dGDP

(Fig. 6b). These stretches are rich in basic residues. The indi-

vidual residues His170, Arg186 and Arg218 also interact with

the phosphates. The planar guanidinium group of Arg176

makes a stacking interaction with the nitrogen base of 8-oxo-

dGDP. Lys297 and Glu242 interact with the sugar and the

base, respectively. Interestingly, the short 144–146 stretch in

domain 1, which is also part of site A, is close to the nucleoside

at site B. Tyr145 of this stretch stacks against the base. Another

residue from domain 1, Asp60, forms a hydrogen bond to a

sugar hydroxyl group. Binding of NDP involves the burial of

133 and 200 A2 of the surface area of domain 1 and trans

domain 2, respectively.

Site C, which spans domain 1 and trans domain 2, overlaps

to an extent with sites A and B, particularly the latter. The

electron densities of the ligand at this site are not as good as

those at the other two. The atoms also exhibit high tempera-

ture factors. However, the densities are good enough to place

the ligand unambiguously. The NMP ligand at the site buries

146–170 and 168–177 A2 of the surface areas of domain 1 and

trans domain 2, respectively. The 141–144 stretch of domain 1

is at the boundary of all three sites. Two residues in this stretch

interact with the sugar at site C (Fig. 6c). Arg175 of trans

domain 2, a residue involved in site B, also interacts with the

sugar. Trp61 of domain 1 and Arg296 of trans domain 2 are

involved in interactions with the phosphate group at site C.

Asp59 of domain 1, which is at the boundary of site A, and

Arg169 of trans domain 2, which forms part of site B, interact

with the base at site C. PPi at site B and the nucleoside

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Figure 7Ligand-induced intermolecular and intramolecular movements. (a) Movement of trans domain 2 with respect to domain 1 owing to the binding ofligands. (b) Ligand-induced movements within domain 1.

monophosphate at site C can coexist. However, the sugar

component of the ligand at site B would have serious steric

clashes with the base component of the ligand at site C. Thus,

site C is unoccupied in crystal 7, in which site B is occupied by

a nucleoside diphosphate.

3.5. Ligand-induced movements

Although the overall molecular structure remains the same

in all crystals, specific movements related to ligand binding are

discernible on close examination. As illustrated in Fig. 7(a),

trans domain 2 rotates by 9.6–11.9� in liganded molecules

(crystals 3, 4, 5, 6 and 7) with respect to its position in the

crystals of the unliganded protein

(crystal 1). When the reaction is

complete, as in crystals 8 and 9,

the rotation is reversed, but not

fully. The angle of rotation of

trans domain 2 in these crystals

with respect to the position in

crystal 1 is 3.4–4.6�.

Movements take place within

the domain as well. For instance,

substantial movements, especially

of aromatic residues, take place at

site A on ligand binding (Fig. 7b).

Tyr101 and Tyr145, the side

chains of which stack on either

side of the base, understandably

move towards the base in the

complexes. Tyr101, in addition,

forms a hydrogen bond to a

phosphate group. Tyr58 and

Lys108 also move towards the

ligand to facilitate hydrogen

bonding. In fact, the peptide

stretches to which these residues

belong move, as a whole, towards

the ligand in the complexes. Here

again, these residues and the

peptide stretches that carry them

do not entirely move back to their

position in the crystals of the

apoenzyme (crystal 1) on

completion of the reaction (crys-

tals 8 and 9).

3.6. Comparison with similarenzymes

The NTD of MsMutT1 has low

sequence identities of 18.3 and

13.2% to EcMutT and HsMTH1,

respectively. The three, however,

have remarkably similar core

structures, with r.m.s.d.s of 1.79

and 1.76 A in C� positions

when the structures of EcMutT (Nakamura et al., 2010) and

HsMTH1 (Svensson et al., 2011) are superposed on that of

MsMutT1-NTD. The structures also exhibit significant differ-

ences in the loops and other structural elements surrounding

the active sites (Fig. 8a). Unlike MsMutT1-NTD, insertions in

one such loop in EcMutT and HsMTH1 and an additional

�-hairpin in HsMTH1 help in the formation of closed pockets

by covering the active site. The site in the MsMutT1-NTD

is relatively open. Furthermore, in the nucleotide complexes

the ligands are located deeper in the pocket in EcMutT and

HsMTH1 compared with that in MsMutT1-NTD (Fig. 8b).

Therefore, there could be hitherto unappreciated differences

in substrate specificities. The core of the NTD of the

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Figure 8Structure of and ligand location in MsMutT1-NTD and its homologues. (a) Structures of MsMutT1-NTD,E. coli MutT (EcMutT), human MTH1 (HsMTH1) and Bifidobacterium adolescentis MutT1 (BaMutT1).Longer loops in all of these, except MsMutT1-NTD, and an additional �-hairpin in HsMTH1 (blue), shownin boxes, cover their active sites. (b) An overlay of ligand-bound structures of MsMutT1 (NTD with ligand),EcMutT (PDB entry 3a6u; Nakamura et al., 2010) and HsMTH1 (PDB entry 3zr0; Svensson et al., 2011),showing the locations of the ligands.

putative MutT1 of B. adolescentis (BaMutT1-NTD), the

structure of which is available in the PDB (PDB entry 3fjy),

has a fold similar to that of MsMutT1-NTD. Here again, a

larger insertion in another loop seems to provide a lid for

what appears by analogy to be the ligand-binding site. A

description of the structure of this protein has yet to be

published.

CTD is structurally similar to several of the branch 1

members of the histidine phosphatase superfamily. In parti-

cular, it is structurally close to the SixAs from E. coli and

Nakamurella multipartitia, the putative SixA from Agro-

bacterium tumefaciens and the alr0221 protein from Nostoc sp.

For instance, the structures of the SixAs from E. coli and

N. multipartitia superpose on the structure of MsMutT1-CTD

with r.m.s.d.s of 2.01 and 1.80 A, respectively. All of these,

along with MsMutT1-CTD, represent structures with the

minimal core of branch 1 of the histidine phosphatase super-

family and differ from the rest of the branch 1 members and all

of the branch 2 members of the histidine phosphatase super-

family in that they lack an extra subdomain that is found in the

latter (Fig. 9). This extra subdomain seems to cover the active

site of these enzymes and helps in the formation of a deeper

pocket. The active sites of SixA and MsMutT1-CTD are

relatively shallow and open owing to the absence of this extra

domain. However, the mycobacterial enzyme exhibits some

similarity to both groups. His170 in MsMutT1-CTD is found at

a homologous position in both of the groups. Asp183 in

MsMutT1-CTD occupies the same spatial location as the

catalytic aspartate residue in the homologues with a shallow

pocket. Glu242, on the other hand, occupies a location similar

to the catalytic glutamate residue in the structural homologues

with a deeper active-site pocket. Both of these residues have

been proposed to act as a proton donor in the phospho-

histidine-mediated phosphatase activity of these structural

homologues. However, the orientation of the nucleoside

diphosphate at site B is not such as to enable catalysis invol-

ving these residues.

MsMutT1 is an example of a protein in which a Nudix

hydrolase domain occurs along with a histidine phosphatase

domain. The crystal structure of only one other protein

containing histidine phosphatase and Nudix hydrolase

domains has so far been determined (PDB entry 3fjy). It may

be mentioned that the histidine phosphatase domain often

occurs along with other domains (Rigden, 2008). The other

domains which are known to occur along with the histidine

phosphatase domain include kinase domains (for example

phosphofructokinase, inositol hexakisphosphate kinase and

Vip1 kinase domains), the 2H phosphoesterase domain,

reductase or dehydrogenase domains, carbohydrate-binding

modules, ankyrin repeats, SH3 (Src homology 3) domains

and the UBASH (ubiquitin-associated and SH3 domain-

containing) domain. In the light of the roles suggested for the

histidine phosphatase domain in many proteins, a role for it

beyond catalysis cannot be ruled out in MsMutT1.

3.7. Insights into the mechanism of action

The biochemical and crystallographic results presented

above lead to a plausible proposal for the mechanism of action

of this unusual enzyme. In most of the crystals obtained under

conditions with a limited supply of magnesium ions, a

magnesium ion coordinated by the main chain of Lys65, the

side chain of Glu85 and water molecules is close to P� of the

NTP (Fig. 10a). The situation is analogous to that in an NTP

complex of EcMutT (Lin et al., 1997), except that the ion in

this case is close to P� and the reaction product of the E. coli

enzyme is a nucleoside monophosphate. One of the water

molecules that coordinate to the magnesium is also close to

the �-phosphate group and could be involved in nucleophilic

attack on P� in an associative mechanism. Glu81, the side

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Figure 9Comparison of MsMutT1-CTD (the CTD of apo MsMutT1) with its structural homologues E. coli SixA (EcSixA; PDB entry 1ujc; Hamada et al., 2005)and Bacillus subtilis PhoE (BsPhoE; PDB entry 1h2e; Rigden et al., 2003), both of which belong to branch 1 of the histidine phosphatase superfamily.MsMutT1-CTD and EcSixA lack an extra subdomain (in blue) that covers the active site in the BsPhoE structure. The conserved histidine residueindicating the location of the active site is shown in pink.

chain of which is hydrogen-bonded to the water molecule,

could act as a general base to deprotonate the attacking water

ligand. The side chain of Arg80 appears to be involved in

maintaining the correct orientation of the glutamyl side chain.

The side chain of Arg55 is positioned appropriately to

protonate the leaving O atom (O3B) and thus might serve as a

general acid. Lys65, Lys67 and Lys108, which are hydrogen-

bonded to the phosphoryl O atoms of NTP, help to hold the

substrate in the appropriate position. Crystal 6 represents a

situation in which the conversion of 8-oxo-GTP to 8-oxo-GDP

is complete but the product is still located at site A.

Long exposure to magnesium ions, as in crystal 7, appears to

have helped to take the reaction to the next step and trans-

locate the NDP from site A to site B. Energetics appear to

favour this translocation. Energy calculations indicate that the

free energies of interaction of the NDP at site A and site B to

be �65.3 and �70.4 kJ mol�1, respectively. From the pictures

at the two extreme positions, it is difficult to work out the path

between the two. However, from their location at site A and

site B in the relevant crystals, Tyr58, Tyr145, Arg169, Gly272

and Lys273 appear to be involved in the translocation of the

NDP from site A to site B.

The next issue to be addressed is the mechanism of

conversion of 8-oxo-GTP or 8-oxo-dGTP to the corresponding

nucleoside monophosphates. The available crystal structures

with a head-to-tail arrangement do not provide an explanation

for the hydrolysis of the NTP to a nucleoside monophosphate

and a pyrophosphate. However, the structure of MsMutT1 in

crystal 2 provides clues as to how catalysis might proceed.

Notably, Glu127 in this crystal moves closer to the substrate

compared with its position in the other crystals. Both of the

side-chain O atoms of this residue, in addition to the main-

chain O atom of Lys65, a side-chain O atom of Glu85 and

water molecules, coordinate to a magnesium ion (Fig. 10b).

One of these is hydrogen-bonded to a water molecule, which is

in a position to attack P�, with the side chain of Glu127 acting

as a general base to activate the water molecule. Lys108 is

appropriately positioned to donate a proton to the leaving O

atom (O3A) and it appears to facilitate the departure of the

NMP product. Lys67, which is hydrogen-bonded to the

�-phosphate and �-phosphate, might facilitate the departure

of pyrophosphate. The proposed mechanism applies equally

well to the conversion of NDP to NMP when the enzyme has a

conformation similar to that found in crystal 2.

The available results lead to a proposal that the arrange-

ment involving the characteristic juxtaposition of domain 1

and trans domain 2, which is observed in all but one crystal,

and that which does not involve such juxtaposition (crystal 2)

contribute to the catalytic machinery of the enzyme. One

could lead to NDP and PO4 as the products, while the other

could help to produce NMP and PPi. The two could be in

dynamic equilibrium in the cellular milieu with the arrange-

ment which brings together domain 1 and trans domain 2 as

the predominant species. As indicated earlier, energy calcu-

lations and the location of relevant residues suggest the

possibility of translocation of NDP from site A to site B. The

NMP in solution produced by the enzyme in the structural

state found in crystal 2 could have been picked up at site C by

the protein in the dominant arrangement found in all other

crystals.

The obvious structural role of the second domain is in

the generation of sites B and C through the head-to-tail

arrangement of MsMutT1 molecules. It also provides a lid to

the relatively open pocket at site A, thus forming a cave-like

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362 Arif et al. � Mycobacterium smegmatis MutT1 Acta Cryst. (2017). D73, 349–364

Figure 10Enzyme-catalysis schemes. Hydrolysis of (a) 8-oxo-GTP to 8-oxo-GDP and a phosphate and (b) of 8-oxo-dGTP to 8-oxo-dGMP and a pyrophosphate.Arrows in red indicate the activation of a water nucleophile with the aid of a general base followed by its attack on the phosphate centres. The arrows inpurple direct the leaving O atom towards proton donors (general acids).

structure. By doing so, it might provide additional stability to

the substrate bound at site A, which would otherwise have its

sugar exposed to the solvent, and thus might aid in efficient

catalysis. Thus, trans domain 2 might, in part, play a role

similar to that played by the extra loop produced by the 3–4-

residue insertions in EcMutT and HsMTH1 and the additional

�-hairpin in HsMTH1 (Mishima et al., 2004). Additionally,

trans domain 2 might be involved in promoting product

release via its interactions with the loops surrounding the

active site. Trigger events, mediated by structural elements

that are not involved in direct interactions with the substrate

or product, have been proposed to facilitate substrate binding

(Ge et al., 2013) and product release (Bailey et al., 2002) in

other Nudix hydrolases. However, considering the many roles

that the histidine phosphatase domain assumes in biological

systems, it is unlikely that its role in MsMutT1 is only as a

structural component of the catalytic machinery. Its additional

role(s), perhaps not necessarily involving catalysis, will

hopefully become clear through further studies of the

enzyme.

The above observations on MsMutT1 are likely to be

relevant to MtMutT1 as well. The enzymes from the two

mycobacterial species exhibit a sequence identity of 60.8%.

Perhaps more importantly, 27 of the 32 residues involved in

interactions with the ligands are identical in the two enzymes.

Two of the remaining residues interact with a nucleotide

through main-chain atoms and a third is only involved in a

water bridge. There are only two differences of consequence,

one of which involves a conservative substitution (Arg to Lys).

Neither of them is involved in the proposed mechanism of

action.

4. Conclusions

MsMutT1, which is made up of a Nudix hydrolase domain

(domain 1) and a histidine phosphatase domain (domain 2),

efficiently converts 8-oxo-GTP and 8-oxo-dGTP to the

respective nucleoside diphosphates. At higher concentrations

the enzyme can also catalyse the conversion of the NTPs to

NMPs and pyrophosphate. The same reaction can be cata-

lysed, albeit much less efficiently, by domain 1 alone, but not

by domain 2. Thus, the role of domain 2 in in vitro experiments

is one of speeding up the reaction.

In eight of the nine crystals studied, one of the apoenzyme

and the others grown under different conditions in the

presence of 8-oxo-GTP and 8-oxo-dGTP, the molecules are

arranged in a head-to-tail fashion, with domain 1 of one

molecule and domain 2 of a neighbouring molecule (trans

domain 2) in periodic proximity. Domain 1 and trans domain 2

together form a functional unit and contain three binding sites.

Site A, which binds NTPs, is almost exclusively made up of

residues from domain 1. Site B, for NDPs or a pyrophosphate,

and site C, for NMPs, are at the interface between domain 1

and trans domain 2, in an unusual case in which binding sites

are produced by intermolecular interactions. The disposition

of residues, a magnesium ion and a water molecule at site A,

and the location of NTP and NDP at the site, lead to a plau-

sible proposal for the mechanism of hydrolysis of 8-oxo-GTP

and 8-oxo-dGTP to the respective NDPs.

The structure of the active site in the lone crystal in which

domain 1 and trans domain 2 are not in proximity is very

similar to that in which they are. In the former, however, a

neighbouring peptide stretch bearing a glutamyl residue

moves close to the ligand. This residue, along with a water

molecule, appears to facilitate the hydrolysis of the NTP to

NMP and pyrophosphate. The more frequently observed

arrangement which leads to the production of NDP and

phosphate and the arrangement that facilitates the hydrolysis

into NMP and pyrophosphate could be in dynamic equili-

brium in the cellular milieu. This proposal is in agreement with

the results of biochemical experiments on MsMutT1.

Acknowledgements

X-ray data were collected at the X-ray facility for Protein

X-ray Structure Determination and Protein Design at this

institute, supported by the Department of Science and Tech-

nology (DST), and at ESRF, Grenoble through an arrange-

ment made by the Department of Biotechnology (DBT). The

Graphics Facility supported by the DBT was used to perform

some of the computations. The DLS and SAXS measurements

were carried out in the laboratory of Dr R. Sankarnarayanan

at the Center for Cellular and Molecular Biology, Hyderabad.

UV is a J. C. Bose fellow of the DST. MV is Albert Einstein

Professor of the Indian National Science Academy. This work

was supported by a research grant from the DBT.

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