Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B12 Romila Mascarenhasa,1, Markus Ruetza,1, Liam McDevitta, Markos Koutmosb,c, and Ruma Banerjeea,2

aDepartment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0600; bDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109-0600, and cDepartment of Biophysics, University of Michigan, Ann Arbor, MI 48109-0600

Edited by Amie K. Boal, Pennsylvania State University, State College, PA, and accepted by Editorial Board Member Stephen J. Benkovic October 20, 2020 (received for review April 16, 2020) Cobalamin is a complex organometallic cofactor that is processed Human and Methylobacterium extorquens (Me) ATR, which have and targeted via a network of chaperones to its dependent been characterized most extensively, also double as chaperones, enzymes. AdoCbl (5′-deoxyadenosylcobalamin) is synthesized transferring AdoCbl directly to MCM (Fig. 1A) rather than re- from cob(II)alamin in a reductive adenosylation reaction catalyzed leasing the high-value product into solution (12–17). Cofactor by adenosyltransferase (ATR), which also serves as an escort, de- loading onto MCM is gated by the GTPase activity of the G livering AdoCbl to methylmalonyl-CoA mutase (MCM). The mech- protein chaperone CblA (18). Despite the overall structural and anism by which ATR signals that its cofactor cargo is ready functional conservation of the cofactor loading processes, there (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. are important differences in regulation between the human (19) In this study, we have obtained crystallographic snapshots that – reveal ligand-induced ordering of the N terminus of Mycobacte- and the better-characterized bacterial systems (15 17). However, rium tuberculosis ATR, which organizes a dynamic cobalamin bind- there is virtually no information on the synthesis of AdoCbl by ing site and exerts exquisite control over coordination geometry, Mtb ATR or its delivery to MCM. reactivity, and solvent accessibility. Cob(II)alamin binds with its The PduO class of ATRs is one of three found in nature, in dimethylbenzimidazole tail splayed into a side pocket and its cor- addition to CobA and EutT, that have arisen via convergent rin ring buried. The cosubstrate, ATP, enforces a four-coordinate evolution (20). The PduO ATRs are homotrimers in which the cob(II)alamin geometry, facilitating the unfavorable reduction to active sites reside at subunit interfaces. The human and Me cob(I)alamin. The binding mode for AdoCbl is notably different ATRs bind AdoCbl in the “base-off” state, which specifically BIOCHEMISTRY from that of cob(II)alamin, with the dimethylbenzimidazole tail refers to the conformation in which the endogenous 5,6-dime- tucked under the corrin ring, displacing the N terminus of ATR, thylbenzimidazole (DMB) base is not the α- or lower axial ligand which is disordered. In this solvent-exposed conformation, AdoCbl to cobalt. The “base-on” to base-off switch increases the redox undergoes facile transfer to MCM. The importance of the tail in potential by ∼100 mV (21) and represents a strategy for easing cofactor handover from ATR to MCM is revealed by the failure of 5′-deoxyadenosylcobinamide, lacking the tail, to transfer. In the the challenge of reducing cob(II)alamin to cob(I)alamin, which ′ absence of MCM, ATR induces a sacrificial cobalt–carbon bond ho- subsequently attacks the C5 -carbon of ATP displacing triphos- molysis reaction in an unusual reversal of the heterolytic chemistry phate (PPPi) and forming AdoCbl (Fig. 1A). It is not known that was deployed to make the same bond. The data support an whether a dedicated or shared reductase supports ATR activity. important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites. Significance

cobalamin | crystal structure | cofactor | trafficking | kinetics Coenzyme B12 (or 5′-deoxyadenosylcobalamin [AdoCbl]) is a cofactor for methylmalonyl-CoA mutase (MCM), which is im- he persistence of Mycobacterium tuberculosis (Mtb), the portant for propionate metabolism in Mycobacterium tuber- Tcausative pathogen of tuberculosis, is due in part to its culosis and for anaplerosis in humans. AdoCbl is synthesized by metabolic agility in adapting to hostile and nutrient-restricted adenosyltransferase (ATR), which doubles as an escort, deliv- host environments. In particular, the glucose-poor environment ering the cofactor to MCM. The mechanism by which this large of phagosomes promotes mycobacterial subsistence on a diet of cofactor is translocated from ATR to MCM is not known. Our host-derived lipids (1). Catabolism of cholesterol and the crystal structures of M. tuberculosis ATR reveal that mobile β-oxidation of odd-chain fatty acids lead to propionyl-CoA (2), loops dynamically create a customized pocket to control co- which can be utilized via the methylcitrate cycle or the vitamin factor coordination and conformation, regulating its reactivity and transportability. These changes also control solvent ex- B12-dependent methymalonyl-CoA pathway (3). Optimal growth on propionate is dependent on both pathways being posure and signaling to MCM when ATR is ready to transfer operational (4). cargo, in a process where the cofactor tail plays an important role in the handover between proteins. Mtb harbors the requisite set of chaperones to support B12- dependent methylmalonyl-CoA mutase (MCM, Rv1492/1493), Author contributions: R.M., M.R., and R.B. designed research; R.M., M.R., and L.M. per- including adenosyltransferase (ATR or MMAB, Rv1314c) and a formed research; R.M., M.R., L.M., M.K., and R.B. analyzed data; and R.M., M.R., and R.B. GTPase (MMAA or CblA, Rv1496) that have human orthologs wrote the paper. (5–7). MCM isomerizes methylmalonyl-CoA derived via car- The authors declare no competing interest. boxylation of propionyl-CoA, to succinyl-CoA (8, 9), and is This article is a PNAS Direct Submission. A.K.B. is a guest editor invited by the susceptible to inactivation by the immunometabolite, itaconyl- Editorial Board. CoA (10). Mtb does not biosynthesize B12 but scavenges it Published under the PNAS license. 1 from its host (11). Once inside, B12 is assimilated into the bio- R.M. and M.R. contributed equally to this work. logically relevant derivatives, methylcobalamin and 5′- 2To whom correspondence may be addressed. Email: [email protected]. deoxyadenosylcobalamin (AdoCbl). This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ATR is tasked with the synthesis of AdoCbl (Fig. 1A), the doi:10.1073/pnas.2007332117/-/DCSupplemental. cofactor for MCM and type II ribonucleotide reductase in Mtb.

www.pnas.org/cgi/doi/10.1073/pnas.2007332117 PNAS Latest Articles | 1of11 Downloaded by guest on September 26, 2021 Fig. 1. Cobalamin binding to and functions of ATR. (A) In the presence of ATP, ATR binds cob(II)alamin in an unfavorable 4-c geometry. Following a one electron reduction, ATR catalyzes the adenosylation of cob(I)alamin to form 5-c base-off AdoCbl (455 nm), which is transferred to the MCM-CblA complex with concomitant hydrolysis of GTP. In the absence of AdoCbl transfer, the newly formed Co-C bond is weakened in the ternary ATR•AdoCbl•PPPi complex leading to a species with an absorption maximum at 440 nm. In the presence of oxygen, Co-C bond cleavage is observed with concomitant formation of hydroperoxyadenosine (Ado-OOH) and cob(II)alamin, with an absorption maximum at 464 nm, which upon reduction, can serve in another cycle of AdoCbl synthesis. Alternatively, further oxidation of cob(II)alamin leads to aquocobalamin, which is released into solution due to its weak affinity for ATR. (B)In- creasing concentrations of ATR were added to cob(II)alamin (50 μM, black) in anaerobic Buffer A at 25 °C in the presence of 5 mM ATP. Spectra were recorded 5 min after each addition (gray lines). Binding of cob(II)alamin to ATR•ATP results in a strong absorption peak at 464 nm (red, final spectrum). (C) The change

in absorbance at 464 nm (in B) versus ATR monomer concentration yielded KD = 0.44 ± 0.08 μM (mean ± SD, n = 3). (D) Increasing concentration of ATR were added to AdoCbl (30 μM, black) in Buffer A at 25 °C and spectra were recorded 5 min after each addition (gray lines). ATR binding resulted in a spectral shift

from 525 nm to 455 nm (red, final spectrum). (E) The change in absorbance at 525 nm (in D) versus ATR monomer concentration yielded KD = 0.92 ± 0.1 μM (mean ± SD, n = 4).

The cobalt-carbon (Co-C) bond in B12 is typically cleaved and ATP involves a chemically expensive SN2 reaction, con- heterolytically in methylcobalamin-dependent methyl transfer suming three high-energy phosphate bonds. Kinetics favor the reactions but homolytically in AdoCbl-dependent isomerase re- direct transfer of the newly synthesized AdoCbl product to MCM actions (22). ATR, in a remarkable display of chemical versa- (Fig. 1A). When this option is unavailable, human ATR reverses tility, catalyzes both types of Co-C bond manipulations in the chemical course and cleaves the Co-C bond homolytically, gen- same active site (19). The synthesis of AdoCbl from cob(I)alamin erating the radical pair, cob(II)alamin and the 5′-deoxyadenosyl

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.2007332117 Mascarenhas et al. Downloaded by guest on September 26, 2021 radical that is quenched by oxygen, forming hydro- peroxyadenosine (Fig. 1A). Under anaerobic conditions, a weakened but intact Co-C bond is seen as evidenced by the formation of a diamagnetic species with absorption maxima at 389 and 439 nm, in contrast to the starting 455 nm, corre- sponding to base-off AdoCbl (19). The presence of PPPi in the ternary product complex is key to facilitating the Co-C bond homolysis step. In its absence, a third route is promoted: That is, loss of AdoCbl into solution, which is augmented by the R186Q patient mutation (19). While the physiological rationale for the sacrificial Co-C bond cleavage is not known, it is speculated to represent a cofactor sequestration strategy since human ATR binds cob(II)alamin 12-fold more tightly than it binds AdoCbl (19). Prior to this study, it was not known whether Mtb ATR is similarly versatile and able to form and cleave the Co-C bond via different chemical routes. In this study, we provide crystallographic glimpses into how ATP tailors the B12 binding pocket in Mtb ATR and controls its chemical reactivity. Unlike all prior structures reported to date, we have captured the disordered-to-ordered transition at the very N-terminal end in ATR, which wrests the DMB tail away from the corrin ring in the cob(II)alamin reactant state. In the product AdoCbl state, the DMB tail changes conformation and is more solvent exposed, likely providing a molecular handle for the direct transfer of cofactor to MCM. The structures also re- veal how the binding energy of ATP is translated into the con- struction of a B12 binding pocket, enforcing the less-favored four-coordinate (4-c) geometry on cob(II)alamin, thereby bringing its reduction potential within reach of biological BIOCHEMISTRY reductants. Results ATP Shifts the Equilibrium from Five- to Four-Coordinate Cob(II) alamin in Mtb ATR. Binding of free five-coordinate (5-c) base-on cob(II)alamin (472 nm) to Mtb ATR•ATP resulted in a signifi- cant increase in absorption and a shift in the λmax to 464 nm, signaling the presence of 4-c base-off cob(II)alamin (Fig. 1B). Fig. 2. ATP and PPPi influence the EPR spectrum of Mtb ATR-bound cob(II) Cob(II)alamin binds weakly in the absence of ATP but tight alamin. (A and B) EPR spectra of cob(II)alamin (100 μM) in Buffer A in the μ binding (KD = 0.44 ± 0.08 μM) is observed in its presence absence (A) or presence (B) of ATR (100- M trimer). The singlets in the high- field region (lines) and the broad peak at ∼2,600 G (arrow) indicate a minor (Fig. 1C). Isothermal titration calorimetry (ITC) yielded a KD for ATP of 8.0 ± 0.9 μM(SI Appendix, Fig. S1A). AdoCbl binding to population of 5-c cob(II)alamin in which axial DMB nitrogen ligand is Mtb ATR similarly signals a coordination state change from replaced by oxygen from H2O. The major population with triplet super- hyperfine structures corresponds to free cob(II)alamin. (C) Addition of 5 mM base-on six-coordinate (6-c) AdoCbl (525 nm) to a base-off 5-c PPPi to the sample in B resulted in an increase in the fraction of 5-c cob(II) species (455 nm) with an isosbestic point at 478 nm (Fig. 1D). alamin with a water ligand indicated by the increase in intensity at ∼2,700 G The binding constant for AdoCbl determined by spectroscopic (arrow) and well-resolved hyperfine singlets in the high field region (vertical (KD = 0.92 ± 0.1 μM) (Fig. 1E) and by ITC (1.27 ± 0.45 μM) (SI lines). (D) Addition of 5 mM ATP to B resulted in a spectrum that is typical of Appendix, Fig. S1B) titration were comparable and similar to the 4-c cob(II)alamin. Two of the three hyperfine coupling constants are value reported for human ATR (0.96 ± 0.31 μM) (19). Like the indicated by vertical lines. human protein, Mtb ATR binds three AdoCbl molecules per homotrimer but with equal affinity. In contrast, Me ATR binds AdoCbl in only two sites simultaneously (15), and like human anisotropy (g = 3.33, 2.50, 1.80) with cobalt hyperfine constants ATR, exhibits negative cooperativity (16, 19). (A = 235, 194, 203 G) that are characteristic of 4-c cob(II) The electron paramagnetic resonance (EPR) spectrum of Co alamin (24). The EPR data reveal that PPPi and ATP signifi- cob(II)alamin exhibits characteristic features that reflect its co- cantly influence cob(II)alamin binding affinity and coordination ordination environment. The eight-line spectrum of cob(II)ala- min (Fig. 2A) results from hyperfine coupling between the geometry. In the presence of PPPi, cob(II)alamin retains the unpaired electron with the cobalt nucleus (I = 7/2), and each line preferred 5-c geometry, with DMB being substituted by an axial is further split into triplets due to superhyperfine coupling with water ligand. In contrast, ATP enforces an unfavorable 4-c ge- the 14N nucleus (I = 1) in DMB (23). In the presence of Mtb ometry. Next, we investigated the structural basis for the heter- ATR (Fig. 2B), the major signal is identical to that of free cob(II) otropic effects of ATP versus PPPi on cob(II)alamin binding. alamin while the minor species with singlets in the high-field region is indicative of base-off 5-c cob(II)alamin bound to Structural Basis of Base-Off AdoCbl Binding. The 1.65-Å resolution ATR in which the DMB nitrogen ligand is replaced by water. In crystal structure of apo-ATR (Table 1) was obtained by molec- the presence of PPPi, Mtb ATR binds cob(II)alamin stoichio- ular replacement using Mtb ATR (PDB ID code 2G2D). Al- metrically (KD ≤ 0.2 μM) (SI Appendix, Fig. S2) and the EPR though ATR is a homotrimer in solution, its structure was solved spectrum (Fig. 2C) reveals the presence of 5-c base-off cob(II) with a single molecule per asymmetric unit (SI Appendix, Fig. alamin (24). Addition of ATP to ATR•cob(II)alamin (Fig. 2D S4A). The 1.5-Å resolution structure of Mtb ATR•AdoCbl and SI Appendix, Fig. S3) leads to a spectrum with a large g showed ordering of the N-terminal residues (13–29), which are

Mascarenhas et al. PNAS Latest Articles | 3of11 Downloaded by guest on September 26, 2021 Table 1. Crystallographic data and refinement for Mtb ATR Mtb ATR complex (PDB ID code)

ATR•Apo (6WGU) ATR•AdoCbl (6WGS) ATR•AdoCbl PPPi (6WGV) ATR•Cob(II) PPPi (6WH5)

Beamline APS, LS-CAT, D APS, GMCA, B APS, LS-CAT, D APS, LS-CAT, D Wavelength (Å) 1.033 1.033 1.033 1.127 Temperature (K) 100 100 100 100 Space group I 4 1 3 2 P 3 2 1 P 3 2 1 C 1 2 1 Cell dimension α, β, γ (°) 90, 90, 90 90, 90, 120 90, 90, 120 90, 90, 119 a, b, c (Å) 130.4, 130.4, 130.4 87.2, 87.2, 46.8 87.6, 87.6, 46.5 108.4, 62.7, 103.1 Resolution (Å) 34.9–1.65 (1.68–1.65) 46.8–1.5 (1.53–1.5) 39.7–2.15 (2.22–2.15) 54.16–1.8 (1.89–1.86)

Rmerge (%) 6.9 (110) 6.9 (129) 17.6 61) 11.1 (75)

Rmeas (%) 7.0(116) 7.2 (141) 19.0 (69) 13.3 (94)

Rpim (%) 1.2 (34) 2.3 (50) 9.7 (41) 4.9 (38) 31.9 (2.4) 16.1 (2.2) 6.6 (2.2) 12.2 (2.3) CC (1/2) 1.0 (0.73) 0.99 (0.76) 0.97 (0.71) 0.99 (0.82) Completeness (%) 99.3 (90.5) 100 (100) 99.1 (93.9) 91.9 (90.6) Multiplicity 31.9 (10.7) 9.9 (7.7) 6.3 (4.1) 6.5 (5.3) No. of reflections 729,029 (10,801) 326,161 (12,456) 71,663 (3,711) 299,258 (12,179) No. of unique reflections 22,879 (1,013) 33,103 (1,609) 11,342 (906) 46,346 (2,310) Overall B (Å2) (Wilson plot) 26.3 19.5 33.5 16.5 Refinement Resolution range 30.74–1.65 43.6–1.5 39.7–2.15 39.8–1.86 No. of reflections (work/test) 22,845/1,208 33,078/1,628 11,338/552 46,298/2,372

Rwork/Rfree (%) 16.1/17.8 15.4/17.1 18.5/23.0 17.6/21.7 No. of atoms Protein 2,403 2,667 2,592 8,508 Water 87 173 15 206

Ligand: B12 NA 178 178 534 Ado NA 31 31 NA PPPi NA NA 13 39 B-factors(Å2) Protein 35.3 27.2 40.8 28.9

Ligand: B12 NA 23.1 33.2 40.2 Ado NA 19.1 33.7 NA PPPi NA NA 64.2 22.8 Water 41.5 40.2 33.2 32.5 RMSD deviations Bond lengths (Å) 0.008 0.011 0.009 0.006 Bond angles (°) 0.822 1.353 1.388 1.018 Ramachandran plot (%) Favored, allowed, outliers 98.7, 1.3, 0 98.3, 1.7,0 98.8, 1.2, 0 98.0, 2.0, 0 MolProbity score (percentile) 0.65 (100th) 0.88 (100th) 0.75 (100th) 0.95 (100th)

unresolved in the apo-ATR structure (Fig. 3A vs. Fig. 3B). also clamped down by multiple hydrogen-bonding interactions AdoCbl sits in a cleft constructed by adjoining subunits and with the side chains of Arg-133 and Asp-167 and the backbone capped on the upper face of the corrin by an N-terminal amides of Phe-71, Lys-115, and Val-118 (SI Appendix, Fig. S4B). β-hairpin from one subunit. The cleft is sealed on the lower or The structure of AdoCbl-bound to ATR suggests the potential α-face by a mobile loop (residues 108 to 123) that straddles the role of ATP (the adenosyl moiety of AdoCbl being a proxy) in C-terminal tail (176 to 188) from another subunit (Fig. 3C). An organizing the B12 binding site. overlay of the apo- and AdoCbl-bound structures (RMSD = 0.30 Å) shows a subtle rearrangement of the mobile (108 to 123) loop, PPPi Induces Co-C Bond Cleavage in ATR-Bound AdoCbl. We recently which repositions Phe-117 and enforces the base-off state in described that human ATR can cleave the Co-C bond homo- AdoCbl (SI Appendix, Fig. S4B). Phe-117 is 3.6 Å away from the lytically (Fig. 1A), generating cob(II)alamin and hydro- cobalt and precludes access of water as a lower axial ligand. On peroxyadenosine when it is unable to transfer AdoCbl to MCM the opposite face, the 5′-carbon of dAdo sits 2.2 Å away from the (19). We investigated whether Mtb ATR exhibits a similar co- cobalt (Fig. 3D and SI Appendix, Fig. S4D), a distance that is factor sequestration strategy. Addition of PPPi to an aerobic ∼0.1 Å longer than in AdoCbl bound to human ATR (19) and sample of Mtb ATR-bound AdoCbl induced a shift in the ab- ∼0.2 Å longer than in free AdoCbl (2.0 Å) (25). The adenine sorption maximum from 455 to 440 nm (SI Appendix, Fig. S5A), ring is roughly perpendicular to the plane of the corrin ring and which we have previously assigned to a weakened Co-C bond interacts via hydrogen bonds with Arg-137, Glu-140, and Arg-141 (19). While no further change was observed under anaerobic from one subunit and Lys-28, Tyr-36, and the backbone of Gly- conditions (SI Appendix, Fig. S5B), a slow decay of the 440-nm 19 from another subunit (Fig. 3C). These interactions help order peak was accompanied by the appearance of new peaks at 355, the N-terminal β-hairpin element and, with the exception of Gly- 466, and 535 nm under aerobic conditions. The spectra were 19 and Tyr-36, are conserved in human ATR. The corrin ring is assigned as a mixture of ATR-bound 4-c cob(II)alamin (466 nm)

4of11 | www.pnas.org/cgi/doi/10.1073/pnas.2007332117 Mascarenhas et al. Downloaded by guest on September 26, 2021 BIOCHEMISTRY

Fig. 3. Structural basis of AdoCbl binding and Co-C bond cleavage. (A, B, and E) Surface representation of the structures of apo-ATR (A), ATR•AdoCbl (B),

and ATR•cob(II)alamin•PPPi (E). The subunits are in blue, gray, and yellow and B12 is in a ball-and-stick display. The progressive ordering of the N-terminal loop is best seen in the blue subunit, which cups B12 in the ATR•cob(II)alamin•PPPi structure. (C) Close-up of the ATR•AdoCbl active site shows that the N-terminal β-hairpin (blue) sits above the 5′-dAdo moiety of AdoCbl (yellow stick display). The mobile loop (108–123) and the C-terminal tail form the floor of

the B12 binding pocket. (D and F) Fo–Fc(2.5σ) simulated annealing omit maps of ATR•AdoCbl without (D) or with (F) PPPi (orange sticks), which binds in the predicted ATP site. The shortest distance between a PPPi oxygen and the C5′ atom in dAdo is 3.2 Å.

and free aquocobalamin (OH2Cbl, 355 and 535 nm). Binding of 5′-carbon of the deoxyadenosine moiety is 3.2 Å from the OH2Cbl to Mtb ATR is weak (KD ∼ 200 μM estimated by ITC), nearest oxygen atom in PPPi and 2.8 Å from cobalt, indi- explaining its presence in solution (SI Appendix, Fig. S5A, Inset). cating that the Co-C bond is broken (Fig. 3F and SI Appendix, In contrast, human ATR stabilizes 4-c cob(II)alamin against Fig. S4E). The DMB tail remains tucked under the corrin in oxidation and retains it in its binding pocket (19). Thus, while the base-off state, as seen in human ATR (26). The solvent Mtb ATR shares the unconventional chemical potential to make accessibility of the corrin in this structure suggests why and break the Co-C bond via different routes, unlike human cob(II)alamin formed following Co-C bond homolysis, is ATR, it does not sequester the resulting cob(II)alamin product susceptible to oxidation, and is released as OH Cbl. The A 2 (Fig. 1 ). structure does not, however, allow us to infer how PPPi The EPR spectrum of cob(II)alamin formed by PPPi-induced labilizes the Co-C bond. Co-C bond cleavage displayed sharp singlets in the high field region (SI Appendix, Fig. S6C) and was similar to 5-c base-off Structural Basis of Cob(II)alamin Binding. Next, the structure of the cob(II)alamin bound to ATR in the presence of PPPi (Fig. 2C). posthomolysis product complex was obtained at 1.86-Å resolu- Structural Basis of Posthomolysis Complex. The 2.15-Å structure of tion (Table 1) by cocrystallizing ATR with cob(II)alamin and ATR•AdoCbl crystals soaked with PPPi (Table 1) revealed a PPPi under anaerobic conditions. The oxidation state and cobalt difference density that could be modeled as PPPi, which is coordination environment in the crystals prior to X-ray data positioned via interactions with Lys-28, Arg-137, and Asp-167 collection were assessed by EPR spectroscopy of ∼500 ATR•- (Fig. 3F). The binding mode of PPPi is similar in the human cob(II)alamin•PPPi crystals (SI Appendix, Fig. S7). The spectra (26) and Mtb ATR active sites with an RMSD of 0.57 Å. A of ATR crystals and of ATR•cob(II)alamin•PPPi in solution magnesium ion is coordinated to two oxygen atoms in PPPi, were comparable, and revealed the presence of 5-c cob(II)ala- two water molecules, and to the side chain of Asn-163. The min with an axial water ligand (27).

Mascarenhas et al. PNAS Latest Articles | 5of11 Downloaded by guest on September 26, 2021 The 1.86-Å resolution ATR•cob(II)alamin•PPPi structure bridging water molecule, and Lys-28 interacts with the backbone was solved with three molecules per asymmetric unit (Table 1) oxygen of Gly-13. The side chains of Val-3, Leu-5, and Ile-8 form with an RMSD of 0.09; the ligands (B12 and PPPi) were each a hydrophobic floor on the α-face of the corrin ring. refined with an occupancy of 1. Fortuitously, a longer stretch of The N-terminal cup enforces a previously unseen cobalamin the N-terminal residues (3 to 28) was ordered in this structure, conformation in which the tail has vacated the active site to creating a cup-shaped cobalamin binding site (Fig. 3E). The accommodate Val-3 and Leu-5 that are positioned above the N-terminal cup is stabilized by numerous polar and nonpolar 108–123 mobile loop (Fig. 4 A and B). The DMB tail extends interactions (Fig. 4A). The propionamide in pyrrole ring B hy- into an ordered hydrophobic groove that is lined on one side by drogen bonds with Thr-6 and the backbone of Ile-8, Thr-10, and Phe-117, Trp-184, and Pro-186 (Fig. 4A and SI Appendix, Fig. Thr-12 interact with the acetamide on pyrrole ring A through a S8). On the opposite side, Pro-88, Pro-89, and Leu-90 flank the

Fig. 4. Structural basis for cob(II)alamin binding and activation. (A) Close-up of the ATR•cob(II)alamin•PPPi active site showing interactions between the 2+ + N-terminal residues (3 to 29), B12 and PPPi, which are bound between adjacent subunits (blue and gray). The purple and green spheres represent Mg and K , respectively. (B) Surface view of the ATR•cob(II)alamin •PPPi structure shows that the ordered N terminus forces the DMB tail into an extended position. (C)A

Fo–Fc omit map of B12, PPPi and water are shown at 2.5 σ.(D) An overlay of ATR•cob(II)alamin•PPPi (blue) and ATR•AdoCbl•PPPi (yellow) reveals differences in the N terminus and the 78–93 loop and the position of the corrin ring in cob(II)alamin (yellow sticks) due to interactions between acetamide group “A” with PPPi and propionamide group “B” with Thr-6. The extended position of the DMB tail (yellow), contrasts with its tucked position in the AdoCbl structure (red).

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.2007332117 Mascarenhas et al. Downloaded by guest on September 26, 2021 DMB moiety, creating a hydrophobic environment. The ribose oxygen is engaged via a hydrogen bond with the backbone of Leu-90. Surprisingly, this mode of binding is reminiscent of that observed in the B12 chaperone CblC (SI Appendix, Fig. S9), which also binds the DMB tail in a hydrophobic pocket away from the corrin ring created by Pro-37, Pro-36, Leu-35, and Leu- 34 (28). New interactions between PPPi and the N terminus are ob- served due to the presence of a potassium ion in addition to the magnesium ion observed previously (Fig. 4A). Mixed metal co- ordination is also observed in Lactobacillus reuteri ATR•cob(II) alamin and attributed to the presence of KCl in the buffer (29). The physiological relevance, if any, of potassium is not known. It is coordinated to two oxygen atoms in PPPi, the side-chain ox- ygens of Asp-167 and the backbone oxygen of Ile-8 (SI Appendix, Fig. S4C). The bond distances (2.7 to 3.1 Å) are typical for co- ordination to potassium (30). PPPi also forms hydrogen bonds to the nitrogen in the acetamide group in pyrrole ring A and the side chains of Lys-28 and Thr-18. An oxygen from the terminal phosphate of PPPi is ∼4.3 Å from the cobalt and 2.7 Å from an ordered water, which is seen in all three subunits. Unexpectedly, the water is positioned 2.9 Å above the pyrrolic N4 nitrogen and is clearly not within coordination distance to the cobalt atom (3.1 Å), which contrasts with clear EPR evidence for water coordi- nation in the crystals (SI Appendix, Fig. S7). We speculate that cob(II)alamin is reduced in the X-ray beam to cob(I)alamin, which prefers 4-c square planar geometry, and that the water is displaced to a holding position from where it returns as an axial ligand upon oxidation to cob(II)alamin. BIOCHEMISTRY An overlay of the ATR structures with AdoCbl•PPPi and cob(II)alamin•PPPi displays subtle changes in the 78–93 loop and in the β-hairpin (Fig. 4D). The hydrophobic environment below the corrin ring is retained in the ATR•cob(II)ala- Fig. 5. Regulation of cofactor translocation from ATR to MCM. (A) AdoCbl min•PPPi structure, impeding access to a water ligand. The transfer from ATR to MCM•CblA•GTP. Transfer of AdoCbl to MCM in the N-terminal cup reduces solvent access to cobalamin, which might presence of CblA leads to a shift from 455 nm (5-c) to 525 nm (6-c) but no be germane to impeding cob(II)alamin translocation to MCM as further change upon addition of PPPi. (Lower left) ATR (15-μM trimer) was μ μ discussed later (Fig. 3B vs. Fig. 3E). loaded with AdoCbl (15 M, Inset) (black) before addition of MCM (30 M), CblA (60-μM dimer) and GTP (1 mM) and incubated for 20 min at 25 °C. Spectra were recorded every minute (gray traces) and the final spectrum is The DMB Tail Facilitates Cofactor Transfer from ATR to MCM. The shown in red. (Lower right) PPPi (5 mM) was added after 20 min and incu- differences in solvent accessibility and the conformation of the bated at 25 °C; the spectrum was recorded after 20 min (red). No spectro- DMB tail in AdoCbl versus cob(II)alamin bound to ATR sug- scopic changes were observed, indicating that AdoCbl had transferred to gested a role for the tail in cofactor transfer from ATR to MCM. MCM. (B) AdoCbi does not transfer from ATR to MCM•CblA•GTP. Subse- To evaluate the role of the tail, the transfer efficacy with 5′- quent addition of PPPi under aerobic conditions leads to Co-C bond ho- deoxyadenosyl cobinamide (AdoCbi), which has a truncated molysis in ATR-bound AdoCbi and to the formation of a mixture of cob(II) μ tail, versus AdoCbl was tested (Fig. 5). AdoCbl loading from alamin and H2OCbl. (Lower left) ATR (15 M trimer) was loaded with AdoCbi ATR to MCM is gated by the GTPase activity of the chaperone (15 μM, Inset) (black) before addition of MCM (30 μM), CblA (60 μM) and GTP CblA and is accompanied by a spectral shift corresponding to a (1 mM) in Buffer A and incubated for 20 min at 25 °C. Spectra were recorded every minute (gray traces) and the final spectrum is shown in red. (Lower base-off (455 nm) to base-off/His-on (525 nm) transition right) PPPi (5 mM) was added after 20 min and incubated at 25 °C; the (Fig. 5A), which is not observed in the presence of the non- spectrum was recorded after 20 min (red). The increase in absorption at hydrolyzable GTP analog, GMPPCP (SI Appendix, Fig. S10A). 466 nm corresponds to cob(II)alamin resulting from Co-C bond homolysis of

To confirm that AdoCbl remained bound to ATR in the pres- AdoCbi bound to ATR and the subsequent oxidation product, H2OCbl ence of GMPPCP but not GTP, PPPi was added to the reaction (355 nm). mixture to induce Co-C bond homolysis. In the presence of GMPPCP (SI Appendix, Fig. S10A), the increase in absorbance at 355 and 535 nm signaled formation of H2OCbl from the ho- (Fig. 5B). Similar results were obtained when GTP was molysis product cob(II)alamin, confirming that AdoCbl substituted with GMPPCP (SI Appendix, Fig. S10B). The absence remained bound to ATR. In contrast, PPPi did not induce a of PPPi induced effects on AdoCbl or AdoCbi bound to MCM spectral shift in the presence of GTP (Fig. 5A), consistent with were independently confirmed (SI Appendix, Fig. S12). the transfer of AdoCbl to MCM. Next, we examined the potential to transfer AdoCbi, which Discussion binds to MCM with a KD value of 5.0 ± 0.2 μM(SI Appendix, Fig. Cobalamin is an essential cofactor and its high value to cells is S11). Interestingly, AdoCbi binding to MCM is not accompanied reflected in the elaborate system of chaperones that has evolved by the expected spectral shift to 525 nm, indicating that the DMB to process and deliver it to target enzymes (5). ATR is one such tail plays a role in positioning the MCM-derived histidine for chaperone that transforms the inactive cob(II)alamin cofactor to cobalt coordination (SI Appendix, Fig. S12). Unlike AdoCbl, the active AdoCbl form and then delivers it directly to MCM cofactor transfer was not observed from ATR to MCM with (15). Cobalamin is large and is typically tethered via a multitude AdoCbi; PPPi-induced Co-C bond homolysis at the end of the of interactions in protein binding pockets. It is not known how its transfer experiment indicating retention of AdoCbi on ATR movement between trafficking proteins is orchestrated nor how

Mascarenhas et al. PNAS Latest Articles | 7of11 Downloaded by guest on September 26, 2021 the process discriminates between the inactive and active co- factor forms. In this study, a series of crystal structures of Mtb ATR have revealed the role of mobile loops in the dynamic construction of the cobalamin binding site and captured the cofactor in two distinct conformations with bound substrate [cob(II)alamin] versus product (AdoCbl). The structures reveal substantial changes that begin with the enthalpically driven binding of ATP, which leads to partial structuring of the N-terminal 13–29 residues into a β-hairpin that has been previ- ously observed in ATR structures with ATP or AdoCbl (19, 26). Architecturally, the β-hairpin serves as the roof of the active site and positions the adenosine moiety (Fig. 3B), while the 108–123 mobile loop together with the C-terminal tail, form the floor of Fig. 6. Model for AdoCbl translocation from ATR to MCM. The N terminus the active site (Fig. 3C). The mobile loop wedges Phe-117 be- of ATR is ordered by ATP into a cup for cob(II)alamin binding and forces the DMB tail into a hydrophobic side pocket. This conformation favors the re- tween the cobalt and the DMB, enforcing a base-off conforma- ductive adenosylation reaction. In the product complex, the first approxi- tion, albeit the DMB tail remains tucked and proximal to the mately eight residues are disordered, which accommodates the tucked-tail corrin as seen previously in human ATR (19). Notably, the DMB conformation and increases solvent accessibility to the cofactor. The histi- tail is exposed to solvent and largely free of interactions with the dine residue on MCM, which serves as the lower ligand to AdoCbl, serves an protein. As discussed below, we posit that this conformation important role in its translocation. signals the “active” transfer-ready form of ATR for loading MCM. Remarkably, in the ATR structure with PPPi and cob(II)ala- min, a more extensive ordering of the N terminus is observed. drives disordering of the N-terminal approximately eight resi- The tip of the N terminus, which is resolved from residue 3 on, dues. In this conformation, the DMB tail is less encumbered by encircles the corrin ring, forcing the DMB tail out into a hy- interactions with the protein and more solvent accessible and drophobic side groove (Fig. 4A). In this conformation, the co- signals to MCM that ATR is ready for cargo delivery (Fig. 6). factor becomes buried and the DMB tail is held via mostly The transfer of AdoCbl to MCM is gated by the GTPase activity hydrophobic interactions. We posit that this conformation sig- of yet another chaperone, CblA (18, 36). nals the presence of “inactive” cob(II)alamin in ATR and dis- The B12 binding domain in MCM is largely preorganized with favors transfer to MCM. The α-face of the corrin remains in a the exception of the loop bearing the histidine residue that serves hydrophobic environment, disallowing coordination by water. In as the lower axial ligand to AdoCbl. Previous studies have shown the presence of ATP, cob(II)alamin becomes 4-c (Fig. 2D), in- that this histidine residue is important for the translocation of dicating that the β-face is devoid of a water ligand, as is also the AdoCbl from ATR to MCM (15). We propose that the histidine case with human ATR (14). In contrast, in the presence of PPPi, residue on MCM and the solvent accessibility of the DMB tail on cob(II)alamin is 5-c, and this “posthomolysis” state is susceptible ATR, facilitate AdoCbl transfer to MCM, resulting in the pre- ferred 6-c (base-off/His-on) geometry (Fig. 6). The model in- to oxidation, leading to OH2Cbl, which is readily lost from Mtb ATR (SI Appendix, Fig. S5A). vokes a role for the DMB tail in translocation between ATR and The switch from base-on to base-off cob(II)alamin in solution MCM, which is supported by the transfer of AdoCbl but not in which the axial nitrogen (DMB) ligand is replaced by oxygen AdoCbi between their active sites (Fig. 5). (H O), increases the reduction potential from −610 mV This model explains several observations. It accounts for the 2 = ± μ to −490 mV versus the standard hydrogen electrode (21). Al- tighter binding of cob(II)alamin (KD 0.44 0.08 M in the ± μ though we do not have an estimate for the redox potential of the presence of ATP) versus AdoCbl (0.92 0.1 M) due to inter- 4-c cob(II)alamin/cob(I)alamin couple, stabilizing 4-c cob(II) actions between the extended DMB tail and residues on ATR. It alamin should render more favorable its reduction to cob(I) also presents a structural basis for discrimination by MCM be- alamin, the supernucleophilic species that attacks ATP to form tween AdoCbl- versus cob(II)alamin-loaded ATR, promoting AdoCbl (Fig. 1A). The physiological reducing partner of ATR is cofactor transfer only from the former. Finally, it identifies an unknown, although flavoprotein oxidoreductases can deliver important role for the DMB tail in cofactor transfer, which might electrons for AdoCbl synthesis in vitro (31). in fact be more generally applicable to other protein partners in The “extended-tail” conformation of cob(II)alamin has not the trafficking pathway. Since the elucidation of the bacterial been previously seen in ATR structures (SI Appendix, Fig. S13), methionine synthase structure, which revealed a base-off/His-on and together with the capture of the “tucked-tail” in the same conformation (37), the rationale for this B12 binding mode has protein is notable for several reasons. B12 enzymes have previ- remained unclear. While the attention has been primarily fo- ously been seen to bind the cofactor either with the tail tucked cused on steric differences between histidine and DMB informed [in the base-on (32–34) or base-off (19) state] or extended away by a wealth of model studies on trans effects, we propose that the from the cobalt ion (10, 28, 35). The dual conformations cap- tail is an instrument used by intracellular trafficking proteins for tured in ATR is unique and leads us to propose a functional role cofactor translocation. for the protein loop and cofactor tail dynamics in both tuning We have considered whether one of the two cobalamin con- chemical reactivity and signaling in the trafficking pathway formations in the Mtb ATR structures is an artifact of the crys- (Fig. 6). In this model, the favorable enthalpy of ATP binding is tallization conditions. This is germane particularly for the associated with stabilizing cob(II)alamin in an “extended-tail” tucked-tail conformation since the human ATR structure conformation. The hydrophobic environment around the corrin where this was previously observed (19) was truncated and the facilitates cob(II)alamin reduction by shifting the redox potential first residue corresponded to Thr-6 in the Mtb ATR. The trun- to within easier reach of biological reducing systems, and signals cation was introduced to facilitate crystallization of human and that the cargo on ATR is in the inactive form and not ready for other ATRs due to the predicted mobility of the N-terminal transfer to MCM. In this state, the N-terminal cup impedes ac- segment (26, 38). We note that the structures reported in this cess of MCM to ATR-bound cob(II)alamin. Synthesis of AdoCbl study with either AdoCbl or AdoCbl and PPPi were obtained triggers tail swapping (i.e., the N-terminal tail vacates the active with full-length Mtb ATR and reveal a remarkably similar site while the DMB tail moves in), although it is unclear what tucked-tail cobalamin conformation. Furthermore, while the

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.2007332117 Mascarenhas et al. Downloaded by guest on September 26, 2021 electron density of the DMB tail in the L. reuteri ATR was dis- degassed under vacuum in the dark at 15 °C using a ThermoVac sample ordered beyond the phosphate group (29), its orientation was degasser. AdoCbi (500 μM) was added to MCM (20 to 22 μM). The data were similar to the extended-tail conformation in our Mtb ATR•co- fitted to a single binding site per MCM heterodimer model using the b(II)alamin•PPPi structure. Based on these arguments, we MicroCal Origin program. conclude that the tucked- and extended-tail conformations that PPPi-Induced Co-C Bond Homolysis in ATR-Bound AdoCbl. ATR (25 μM trimer) we have fortuitously captured are functionally relevant to ATR. and AdoCbl (25 μM) were incubated in Buffer A for 5 min at 25 °C. Then, The residues corresponding to Ala-2, His-4, Leu-5, and Ile-8 that sodium triphosphate (1 mM) was added and the absorption spectra were cup the α-face of the corrin ring in Mtb ATR are similar but not recorded every min for 20 min. The same reaction was also monitored in an

identical to the corresponding residues in human ATR (Thr, anaerobic chamber (O2 < 0.3 ppm), using anaerobic solutions. Arg, Ile, and Ile), while residues 8 to 14 in the Mtb sequence that contribute to the PPPi binding site are conserved, with the ex- Transfer of AdoCbl or AdoCbi from ATR to MCM. ATR (18.75 μM trimer) was ception of Arg-11, which is replaced by a lysine in the mixed with 18.75 μM AdoCbl or AdoCbi in 160 μL Buffer A and incubated at human sequence. 25 °C for 15 min. Then, 40 μL of a premixed solution of 150 μM MCM (dimer), 300 μM CblA (dimer), and 5 mM GTP or GMPPCP was added. The final In summary, we have captured multiple poses of the N ter- μ μ μ μ minus of Mtb ATR, which are correlated with the identity of the concentrations were: 15 M ATR, 15 M AdoCbl, 30 M MCM, 60 M CblA, and 1 mM GTP or GMPPCP. Spectra were recorded every minute for 20 min. bound substrates and accommodate two distinct conformations At the end of the reaction, PPPi was added to a final concentration of 5 mM of the cofactor tail. In combination with the biochemical data, we and spectra were recorded every minute for 20 min. have demonstrated that protein loop movements create a flexible binding site that sequesters and enhances cob(II)alamin reac- AdoCbi Synthesis. AdoCbi was prepared under dim light by cereous hydroxide tivity and subsequently increases solvent exposure of the AdoCbl hydrolysis of AdoCbl by modification of the published procedure (40). Briefly, product, promoting its translocation to MCM. We posit that the NaOH (220 mg, 5.5 mmol) was dissolved in 23 mL of H2O followed by the DMB tail may be generally important in the movement of the addition of cerium nitrate hexahydrate (1.2 g, 2.7 mmol). A solution of μ large cobalamin cofactor between proteins involved in the AdoCbl (100 mg, 63 mol in 2 mL of H2O) was added to the cereous hy- trafficking pathway. droxide suspension, and the reaction mixture was heated to 100 °C with vigorous stirring. After 1 h, the reaction was cooled to room temperature, Materials and Methods and the pH was adjusted to 8.5 with concentrated ammonium hydroxide. The mixture was centrifuged for 15 min at 2,236 × g. The supernatant was Materials. All reagents used were purchased from Sigma Aldrich unless in- decanted, and the residue was washed twice with 10 mL of water. The dicated otherwise. Tris(2-carboxyethyl)phosphine (TCEP) was from GoldBio. supernatants were pooled, and the solution was desalted using an RP-18 cartridge (Waters). The cartridge was washed with 10 mL of water and the BIOCHEMISTRY Mtb Cloning, Expression, and Purification of B12 Enzymes. Recombinant Mtb product was eluted with methanol. The solvent was removed by lyophili- MCM, CblA, and ATR were purified exactly as reported previously (10). zation. Further purification was achieved by preparative HPLC on an RP-18 Buffer A (50 mM Hepes, 150 mM KCl, 2 mM MgCl2, 2 mM TCEP, 5% glycerol column (Luna C-18 250 × 10 mm; Phenomenex) with the following solvent adjusted to pH 7.5 with 10 M KOH) was used unless otherwise noted. system: Solvent A: 10 mM phosphate buffer, pH 6.5; solvent B: acetonitrile; 0 to 2 min, 2% B isocratic; 2 to 12 min, 2 to 15% B; 12 to 25 min, 15 to 18% B; AdoCbl Binding to ATR. The binding affinity of AdoCbl to Mtb ATR was de- 25 to 30 min, 18 to 40% B; 30 to 33 min, 40 to 60% B; 33 to 40 min, 60% B termined in one of two ways. In the first method, binding was monitored by isocratic; 40 to 43 min, 60 to 2% B; 43 to 47 min, 2% B. Fractions containing electronic absorption spectroscopy of a solution containing AdoCbl (50 μM) AdoCbi were desalted using an RP-18 cartridge. The solution was lyophilized in 200 μL of Buffer A. Aliquots of ATR were added and the change in ab- to obtain 51 mg of AdoCbi (64% yield) as an orange powder. sorbance at 525 nm was recorded following a 10-min incubation at 25 °C. The data were fitted to a single binding site/ATR monomer using Dynafit EPR Experiments. EPR spectra were recorded on a Bruker EMX 300 equipped (39). In the second method, ITC analysis was performed using ATR (20 to with a Bruker 4201 cavity and a ColdEdge cryostat. The temperature was μ 25 M trimer) at 20 °C in Buffer A in a 1.43-mL chamber and injecting AdoCbl controlled by an Oxford Instruments MercuryiTC temperature controller. EPR μ μ (10- L aliquots of 500 to 600 M). Samples were degassed under vacuum in spectra were recorded at 80 K using the following parameters: 9.38-GHz the dark prior to use at 15 °C using a ThermoVac sample degasser. The data microwave frequency, power 2 mW, modulation amplitude 10 G, modula- were fitted to a one binding site per monomer using the MicroCal tion frequency 100 kHz, 3,000 G sweep width centered at 3,500 G, conver- Origin program. sion time 164 ms, time constant 82 ms. Five scans were collected per measurement. EPR simulations were performed using the EasySpin Cob(II)alamin Binding to ATR. Binding of cob(II)alamin to Mtb ATR was program (41). monitored in the presence of ATP or PPPi. In the first method, ATR (5-μM Samples were prepared in an anaerobic chamber as follows. Mtb ATR (100- μ aliquots) was added to a solution of cob(II)alamin (50 M) and ATP (5 mM) in μM timer) was mixed with cob(II)alamin generated in situ from OH2Cbl (100 < Buffer A at 20 °C in an anaerobic chamber (O2 0.3 ppm) and the absorption μM) in 50 mM Hepes, 150 mM KCl, 2 mM MgCl2, 2 mM TCEP, 10% glycerol, spectrum was recorded 5 min after each addition. The change in absorbance pH 7.5 in a total volume of 1.3 mL and incubated for 15 min. The sample as at 464 nm was plotted versus ATR concentration and the data were fitted to split into three 380-μL aliquots. To sample A, 20 μL of buffer was added, to a single binding site model using Dynafit. In the second method, ATR (0- to sample B, 20 μL of 100 mM PPPi, and to sample C, 20 μL of 100 mM ATP was 500-μM monomer) was added to a solution of cob(II)alamin (100 μM) and added to give a final concertation of 5 mM PPPi and ATP. After 15-min in- 10 mM PPPi in Buffer A containing 10% glycerol inside an anaerobic cubation, the samples were transferred into EPR tubes and frozen in liquid chamber. Each sample was incubated for 30 min at room temperature and nitrogen. The free base-on cob(II)alamin control sample was prepared in then transferred into an EPR tube and frozen. EPR spectra were recorded as anaerobic Buffer A containing 10% glycerol, and the base-off sample in described below. The peak area from 2,550 to 2,780 G was integrated and its Buffer A was adjusted to pH 2.0 with concentrated HCl. dependence on the concentration of ATR was plotted. The data were fitted To monitor PPPi-induced Co-C bond cleavage in ATR-bound AdoCbl, the to a one-site binding model using Dynafit. The titrations were repeated in samples were prepared under aerobic conditions as follows: AdoCbl (100 duplicate. μM) was mixed with ATR (100-μM trimer) and incubated for 15 min; then, PPPi (5 mM) was added and incubated for additional 15 min before trans- ATP Binding to ATR. ITC experiments were performed using ATR (15- to 20-μM ferring the sample into an EPR tube and freezing in liquid nitrogen. trimer) at 15 °C in Buffer A in a 1.43-mL chamber and injecting ATP (5-μL EPR spectra of ATR•cob(II)alamin•PPPi crystals were recorded on ∼500 aliquots of 900 to 1,000 μM). Samples were prepared by filtration through a crystals that were transferred into a vial containing the well solution (30% 0.2-μm filter and then degassed under vacuum at 15 °C using a ThermoVac PEG 3350, 0.1 M bis Tris pH 6.5, 5% glycerol). The sample was centrifuged for sample degasser. The data were fitted to a one binding site per trimer using 3 min at 13,604 × g at 4 °C. The supernatant was decanted, and the pro- the MicroCal Origin program. cedure was repeated again to separate precipitate, protein or free cob(II) alamin from solution. The crystals were then resuspended in 300 μL of well AdoCbi Binding to MCM. ITC experiments were performed at 20 °C in Buffer A solution containing 20% glycerol (25% final concentration), transferred into using a 300-μL injection syringe and a 1.43-mL injection cell. Samples were an EPR tube and flash frozen in liquid nitrogen. After recording the

Mascarenhas et al. PNAS Latest Articles | 9of11 Downloaded by guest on September 26, 2021 spectrum, the sample was thawed and the crystalline suspension transferred The Mtb ATR structures were solved by molecular replacement using to a sample tube and centrifuged for 5 min. The supernatant was trans- Phaser (44) in the CCP4 program suite (45). The initial search model was a ferred into a clean EPR tube, and the spectrum recorded. monomer of a previously deposited structure of Mtb ATR (PDB ID code 2G2D). Iterative rounds of model building were performed with COOT (46) Crystallization of ATR. Mtb ATR in 50 mM Hepes pH 7.5, 150 mM KCl, 2 mM and initial restrained refinement in Refmac (47) using isotropic individual B

MgCl2, and 2 mM TCEP was used for screening crystallization conditions factors. Subsequent refinement was performed in Phenix.refine (48). Re- using the sitting-drop vapor diffusion method at 20 °C. Apo-ATR crystals finement included individual B-factor adjustment with maximum-likelihood with the best morphology were obtained with 1.75% PEG 400, 1.5 M am- targets and coordinate minimization. After the complete protein model and monium sulfate, 100 mM sodium acetate/acetic acid pH 5.5, using a 2:1 water molecules were assigned, ligands were added to the model to fit mixture of protein (15 mg/mL) to well solution in a 0.9-μL drop. ATR•AdoCbl difference density and further refined. Chemical restraints for ligands were (1:1.2) cocrystals (15 mg/mL) were obtained from a 2:1 mixture (protein:well generated using eLBOW. After addition of ligands and metals, Ready Set solution) in a 0.9-μL drop from a solution containing 400 mM sodium was utilized to generate metal edits and link files before subsequent re- phosphate monobasic/1,600 mM potassium phosphate dibasic, 100 mM im- finement. The geometric quality of the model was assessed in MolProbity idazole/HCl pH 8, and 200 mM sodium chloride. (49). Data collection and final refinement statistics are summarized in Ta- ble 1. Structural analysis and alignments were performed in PyMOL (50) and Crystals of ATR•AdoCbl (15 mg/mL) also appeared in a solution containing final figures were made using University of California, San Francisco 26% PEG 3350, 0.1 M bisTris pH 6.5, 5% glycerol, in a 3-μL drop (2:1 pro- Chimera (51). tein:well solution), were transferred to a well solution substituted with 5 mM PPPi and soaked for 45 min in a dark room. Crystals of ATR•cob(II) Data Availability. All data are available in the main text or supplementary alamin•PPPi (15 mg/mL) were grown in an anaerobic chamber (<1 ppm O ) 2 materials. The structure factors and coordinates have been deposited in the in a 6-μL drop (1:1 protein:well solution) containing 30% PEG 3350, 0.1 M bis Protein Data Bank, www.wwpdb.org [PDB ID codes 6WGU for Mtb apo-ATR, Tris pH 6.5, 5% glycerol. Prior to setting up crystals, 0.7 mM Mtb ATR in 6WGS for ATR•AdoCbl, 6WGV for ATR•AdoCbl•PPPi, and 6WH5 for 50 mM Hepes pH 7.5, 150 mM KCl, 2 mM MgCl , and 2 mM TCEP was mixed 2 ATR•cob(II)alamin•PPPi]. with 5 mM PPPi, 0.7 mM cob(II)alamin and 4 mM TCEP. ATR•cob(II)ala- min•PPPi crystals were harvested under aerobic conditions. For data collec- ACKNOWLEDGMENTS. This work was supported in part by National Insti- tion, crystals were soaked in a cryoprotectant solution (well solution, 20% tutes of Health Grant R01-DK45776 (to R.B.); American Heart Association [vol/vol] glycerol) and flash-cooled in liquid nitrogen. Grant 19POST34370113 (to R.M.); and National Science Foundation Grant NSF-CHE 1945174 (to M.K.). This research used resources at the Advanced X-Ray Data Refinement. Datasets for apo-ATR, ATR•AdoCbl•PPPi and ATR•- Photon Source, operated for the US Department of Energy Office of Science cob(II)alamin•PPPi were collected at the LS-CAT 21-ID-D, Advanced Photon by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use Source, Argonne National Laboratory using a Dectris Eiger 9M detector at of the LS-CAT Sector 21 was supported by the Michigan Economic Develop- ment Corporation and the Michigan Technology Tri-Corridor (Grant 100 K. Datasets for ATR•AdoCbl were collected at GMCA, 23-ID-B, Advanced 085P1000817). General Medical Sciences and Cancer Institutes Structural Photon Source, Argonne National Laboratory using a Dectris Eiger 16M Biology Facility at the Advanced Photon Source has been funded in whole or detector at 100 K. Data were processed with Xia2 DIALS (42), except for in part by the National Cancer Institute (Grant ACB-12002) and the National ATR•cob(II)alamin•PPPi, which was processed in autoPROC (43). Institute of General Medical Sciences (Grant AGM-12006).

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