Structure of the C-terminal domain of ArfA
The C domain of wild-type ArfA folds into four β-strands and four α-helices, arranged in the topological order αβαβαβαβ (). Three parallel (β1, β2, β3) and one antiparallel (β4) β-strands form a four-stranded β-sheet (β1–β4-β2–β3) that packs against three α-helices (α1, α2, α3), while a fourth helix (α4) extends from the N-terminus of β4. A disulfide bond between C208 and C250 connects the N-terminus of α1 to the C-terminus of α2. All ArfA orthologs from mycobacteria, as well as from Kribbella
and α-proteobacteria, have Cys residues at similar positions, suggesting that the disulfide bond is conserved in all their structures 15
Structures and backbone dynamics of wild-type ArfA-c and ArfA-c(D236A)
The disulfide bond stabilizes the structure (see below) but is not required to maintain the overall fold, and the protein can be reversibly reduced and oxidized by adding reducing DTT (dithiotheritol) or oxidizing GSSG (glutathione disulfide). Indeed, the 1
N NMR spectra obtained after treatment with DTT or GSSG show only minor changes in peaks from residues located near C208 and C250, while peaks from the rest of the protein are not affected (Fig. S1a
. The structure is held together by a network of hydrophobic contacts among side chains in α1, α2 and β4 (L211, I215, V243, L247, Ile323 and V325) and is further stabilized by a hydrogen bond between the backbone amide of V325 and the side chain carbonyl of Q212.
The absence of NMR peaks from residues G226 to E238 in the loop linking β1 to α2 suggests that the loop undergoes conformational exchange between two or more states on an intermediate timescale relative to NMR, and precluded complete structure determination. Although several of these peaks manifest upon acidification (Fig. S1b
), they remain significantly broadened, and no 1
H NOEs can be observed to connect residues G226 to E238 with other polypeptide sites, resulting in significant conformational heterogeneity in the β1-α2 loop. This is in contrast to a recent structural study, which also noted the pH dependence of these peaks but, nevertheless, reported a hybrid pH7.2 / pH3.5 solution structure similar to ours, albeit with a fully ordered loop 11
Upon closer examination of wild-type ArfA we noted that a negatively charged residue (D236) at the start of helix α2 clashes with several hydrophobic residues in the β1-α2 loop (L232), in α2 (L240, A244), in α3 (V281, L285), and in β1 (I223, F225). Indeed, when we mutated D236 to Ala, all of the β1-α2 loop peaks were present at neutral pH (Fig. S2a
), and several NOEs connecting the loop to α3 and to the β-sheet could be measured, enabling us to determine a high resolution structure that is very well defined over the entire length of the protein sequence (). In the structure of the D236A mutant, A236 packs comfortably against L232, I233, L240 and V281, confirming that the negative charge of D236 disrupts this hydrophobic cluster in the wild-type protein at neutral pH, leading to conformational heterogeneity in the β1-α2 loop. Furthermore, the N-terminus of α2 in the mutant is longer by one turn and the β1-α2 loop forms a flap, hinged at the C-terminus of β1 and the N-terminus of α2, that folds over the β-sheet and closes at α3 ().
pH-Dependent Conformational Disorder in the β1-α2 Loop
To determine whether the β1-α2 loop adopts a similar, albeit dynamically disordered, conformation in wild-type ArfA at pH7, we mutated L232, in the middle of the loop, to Gly, and examined its effects on other structured regions of the protein by mapping the peak changes in the 1
N NMR spectrum of ArfA(L232G). The L232G mutation in the loop causes several significant (≥ 0.03 ppm) changes in peaks from residues in α3 and in the β-sheet (Fig. S2b
). In ArfA(D236A), the loop packs against the β-sheet and the presence of strong NOEs establishes close contacts between the methyl protons of L232 and the amide protons of A224 and V281. Since the peaks from A224 and V281 are significantly perturbed by the L232G mutation, we conclude that the loop of wild-type ArfA-c is not dissociated from the rest of the polypeptide at pH7 but maintains close contact with the globular structure, notwithstanding a significant degree of conformational exchange.
To examine backbone dynamics, we measured heteronuclear 1
N NOEs for both wild-type and D236A mutant ArfA-c, at neutral and acidic pH (). The dynamics of isolated ArfA-c are similar to those observed in the connected B and C domains 12
. For both wild-type protein and D236A mutant, negative NOEs, reflecting rapid (ps-ns) backbone motions, are observed at the N-terminus corresponding to the flexible inter-domain linker. Beyond the N-terminus, both wild-type ArfA-c at acidic pH, and D236A mutant at neutral pH, exhibit very similar, positive values (~0.8) of the 1
N NOE, reflecting a rigid backbone for the entire protein sequence. Slightly, but visibly lower 1
N NOEs for residues in the loops (β1-α2; β2-α3; β3-α4) indicate the presence of some restricted motions and may reflect the importance of the loops in protein/ligand interactions.
To further dissect the stabilizing effects of acidic pH and of the C208–C250 disulfide bond, we performed DSF (differential scanning fluorimetry) studies comparing the thermal stability of wild-type and mutant ArfA, under various conditions of pH and oxidation state. After treatment with DTT, ArfA-c undergoes thermal unfolding at 60.1°C, while disulfide bond formation by oxidation with GSSG has a significant stabilizing effect and increases the melting temperature to 63.2°C (). A similar effect is observed for the joint B and C domains (ArfA-bc) where the disulfide bond provides about 1°C of stabilization (), while the melting temperature of ArfA-b, which does not contain Cys, is unaffected by oxidation state (). Notably, ArfA-b has a significantly higher melting temperature (67.4°C) than either the C or combined BC domains, reflecting its overall greater content of secondary structure relative to loops 12
Differential scanning fluorimetry traces showing the effect of the disulfide bond, pH, and D236A mutation on the thermal stability of ArfA
Lowering the pH from 7 to 4 further increases the temperature of thermal unfolding of ArfA-c by 2°C (from 63.2°C to 65.0°C; ). The effect of pH is mirrored by the D236A mutation, which increases the unfolding temperature of ArfA-c at pH7 by a similar amount (from 63.2°C to 65.2°C; ). We conclude that the stabilizing effect of acidic pH on the conformational exchange of the β1-α2 loop of ArfA-c is primarily through neutralization of the negative charge of D236, which enables α2 and the loop to pack against α3 and the β-sheet. Whether this pH-dependent conformational heterogeneity correlates with the pH-dependent physiological function of ArfA is not known.
ArfA binds M. tuberculosis peptidoglycan
While the structure of M. tuberculosis
ArfA-b was unprecedented in the database, ArfA-c shares the same βαβαβαβ core structure and significant amino acid conservation with other domains of the OmpA-like family (Fig. S3
), some of which have been shown to bind peptidoglycan 21; 22; 23
. To determine whether ArfA also shares this function we tested its ability to associate with intact peptidoglycan isolated from M. tuberculosis
The peptidoglycan of M. tuberculosis
() is composed of linear chains of GlcNAc (N-acetyl-D-glucosamine) and Mur (muramic acid) that can be both N-acetylated (MurNAc) and N-acylated with glycolic acid (MurNGlyc) 1; 2; 19; 20
. The sugars are substituted with a heavily cross-linked peptide, containing L-Ala, D-γ-Glu, m-DAP (meso-diaminopimelate), and D-Ala, similar to the composition found Gram-negative bacteria (e.g. E. coli
) and Gram-positive Bacillus
, but distinct from the peptidoglycan that is more typically found in Gram-positive bacteria, where m-DAP is usually replaced by L-Lys. OmpA-like proteins have been identified exclusively in organisms with DAP-type peptidoglycan 17
, suggesting specificity for DAP in their structures.
Chemical structures of (a) M. tuberculosis peptidoglycan and (b) the peptidoglycan biosynthesis intermediate, UMDP
Incubation of soluble ArfA polypeptides with insoluble polymeric M. tuberculosis peptidoglycan caused a significant amount of ArfA-bc, ArfA-c and ArfA-c(D236A) to separate with the insoluble fraction after centrifugation, while all three proteins remained in the supernatant in the absence of peptidoglycan (). In contrast, ArfA-b2 (residues 73–197) remained primarily in the supernatant both in the presence and absence of peptidoglycan. We conclude that ArfA binds polymeric M. tuberculosis peptidoglycan via its C domain. No appreciable difference (within the limits of this pull-down assay) was detected between the peptidoglycan-binding abilities of wild-type ArfA-c and ArfA-c(D236A), indicating that the mutant is active in this respect and that conformational heterogeneity in the β1-α2 loop does not affect peptidoglycan-binding.
Association of ArfA-c with peptidoglycan
Peptidoglycan Recognition by ArfA
To further characterize the peptidoglycan binding site of ArfA we examined its interaction with a soluble intermediate of peptidoglycan biosynthesis, UMDP (also known as Park's nucleotide, ), similar to the peptide found in association with H. influenzae
. Addition of UMDP to 15
N-labeled ArfA-c(D236A) produced several peak frequency and intensity changes in the 1
N NMR spectrum of the protein (). The changes are specific as they map to a surface cavity formed by residues in the β1-α2 and β2-α3 loops, the N-terminus of helix α3 and the C-terminus of helix α4 (), similar to the binding site identified for Pal 21
. Similar effects of UMDP were observed for wild-type ArfA-c at both pH7 and pH4. This is consistent with the observation that both wild-type and D236A mutant ArfA-c bind intact peptidoglycan (), indicating that the acid-dependent structural heterogeneity of the β1-α2 loop does not affect peptidoglycan binding by ArfA. The most striking peak perturbations are observed for residues with high sequence conservation in the OmpA-like family (D228, A230, T261, D262, N263, T264, G265, N270, R277, I280, T297, S302, R319). Notably, mutations of residues corresponding to T261, D262, R277, and R319 all render the OmpA-like protein MotB nonfunctional 24
, underscoring their importance.
Peptidoglycan binding site of M. tuberculosis ArfA-c(D236A)
The interaction is specific for m-DAP at position 3 of the peptide stem, since addition of the L-Lys analog UMKP, more typical of Gram-positive bacteria but absent from M. tuberculosis
, had no effect on the spectrum of ArfA-c (Fig. S4b
), and neither did addition of the dipeptide GMAG, lacking m-DAP (S4c
). Thus, the presence of MurNAc alone is insufficient for binding while m-DAP is required, although we cannot exclude the possibility that MurNGlyc, which is abundant in M. tuberculosis
, may be important. Finally, addition of UMDP had no effect on the spectrum of ArfA-b (Fig. S4d
), confirming that ArfA binds peptidoglycan solely through its C domain.
Unlike the Pal study, where NOEs connecting the peptide to the protein enabled the structure of the complex to be determined 21
, the interaction of ArfA-c with UMDP did not yield any intermolecular NOEs. However, a number of key experimental observations, described below, enabled us to construct a structural model that sheds light on peptidoglycan recognition by ArfA ().
Peptidoglycan recognition by ArfA-c
Although the affinity of ArfA-c for polymeric M. tuberculosis peptidoglycan is strong (enough to precipitate the protein), its binding affinity for monomeric UMDP is weak, and a dose-response experiment, performed by mapping NMR peak changes upon titration of UMDP into the protein, yielded a Kd in the range of 1 mM. This high-specificity, low-affinity interaction suggests that electrostatics (hydrogen bonds, charge-charge contacts) play an important role in mediating UMDP recognition. A prominent surface electrostatics feature of ArfA-c is the presence of two juxtaposed clusters of positive charge at or near the peptidoglycan binding site, formed by R277, R319 and R320 (). In the free protein, these arginines participate in electrostatic interactions within a hydrophobic environment, reflected in the presence of side chain guanidinium NHε peaks, with characteristic downfield chemical shift (e.g. 8.06 ppm for R320), in the NMR spectrum. Indeed, the R320 guanidinium group has polar interactions with E322 and N304, and its hydrocarbon side chain is stabilized by hydrophobic contacts with the side chains of I306 and Y260, all established by NOE connections. The R277 side chain is stabilized by hydrophobic contacts with the aromatic ring of F225, with L271 and I280, all residues with very high OmpA-like conservation. The heteronuclear 1H/15N NOEs measured for the guanidinium NHε peaks of these arginines (0.58 for R277; 0.62 for R319; 0.86 for R320) reflect a rather rigid side chain for R320, but significantly more flexibility for R277 and R319.
The R277 and R319 side chains are positioned to facilitate the formation of a hydrogen bond network that includes their guanidinium groups as well as the hydroxyl of T261, the carboxylate of D262, and the carboxamide of N270. These residues all have NMR peaks that exhibit significant changes in the presence of UMDP: the side chain NHε peaks from R277 and R319, but not R320, undergo substantial shifts, and the side chain NH2 peak of N270 is broadened beyond detection, suggesting a direct interaction with the peptide (). Furthermore, the backbone amide peaks of N270, T261 and D262 are all affected by UMDP. The five T261, D262, N270, R277 and R319 side chains all converge at the peptidoglycan binding site where they provide an effective recognition site for m-DAP (), possibly facilitated by the side chain flexibility of R277 and R319. Peptidoglycan recognition proteins of the immune system rely on a similar, buried electrostatic interaction between conserved Arg and the m-DAP carboxyl group to select for Dap-type peptidoglycan 25
, suggesting that this may be a conserved interaction in a variety of protein folds.
To confirm the identity of the peptidoglycan recognition site, we generated a mutant of ArfA-c(D236A) where R277 was replaced by Glu, and tested its ability to bind UMDP by NMR spectroscopy. Although the R277E mutation alone caused several peak changes compared to the spectrum of ArfA-c(D236A), the perturbations were largely localized to sites near residue 277, the peak movements could be easily tracked, and the overall appearance of the spectrum was maintained, indicating that the overall fold of the protein is preserved. Most importantly, no changes were observed in the spectrum of the R277E mutant upon addition of UMDP (Fig. S5
), indicating that the protein has lost its ability to bind peptidoglycan. Thus, we conclude that R277 is a key residue for the recognition of peptidoglycan by ArfA.
Our model indicates that the side chain of m-DAP is stabilized by charge-charge interactions between its electronegative carbonyl group with the R277 and R319 guanidinium groups and with the N270 carboxamide, and between its electropositive amino group with the D262 carboxyl and the T261 hydroxyl. These residues are strictly conserved in the OmpA-like family and our data show that they constitute the specific recognition site for DAP-type peptidoglycan (). For example, in the structure of H. influenzae Pal, D71 (corresponding to ArfA D262) forms an electrostatic interaction with the side chain NH3 group of m-DAP. Our model also indicates that UMDP could further interact with ArfA-c through contacts of its γ-Glu3 and Ala2 backbone amides with the side chain hydroxyl of S266, of its MurNac OH4 with the backbone carbonyl of G265, of its MurNac O3 with the amide proton of E267, and of its MurNac NH2 with the E267 carboxyl.
Saturation transfer difference NMR experiments show that the amide proton of S302, located in the β3-α4 loop, is in some proximity to the methyl protons of the UDMP MurNAc group (Fig. S6
). The structural position of S302 is identical to that of S208 in H. pylori
MotB, where it appears to stabilize the interaction with a single MurNAc sugar molecule, by forming hydrogen bonds between its side chain hydroxyl group and the O1 atom of MurNAc and between its backbone amide NH and the acetamido oxygen atom (O7) of MurNAc 22
. S302 may assist the interaction of ArfA with peptidoglycan in a similarly way. A Ser or other hydroxyl-bearing residue at this position is conserved in many of the OmpA-like sequences with the exception of YiaD, where it is replaced by Pro. In all sequences, this residue is preceded by a perfectly conserved Gly, which could confer structural flexibility in the loop to further assist interaction with the peptidoglycan.
The highly specific albeit weak interaction of ArfA-c with UMDP is reminiscent of the immune response peptidoglycan recognition proteins, which achieve selective recognition of peptidoglycan type by specifically discriminating for either DAP or Lys in the third position of the peptide stem, and for the degree and type of peptide stem cross-linking 26
. We note that UMDP is chemically and structurally different from polymeric M. tuberculosis
peptidoglycan, whose affinity for ArfA is sufficiently strong to precipitate the protein (). The interaction of ArfA with polymeric peptidoglycan may be strengthened by the highly cross-linked peptidoglycan structure, which could provide additional binding sites for ArfA (e.g. at peptide stem cross-links), cause an effective increase in protein concentration by sterically constraining its diffusion, and reduce the flexibility and conformational space of the peptidoglycan peptide stem. These effects could be particularly important for M. tuberculosis
where 75% of peptidoglycan is cross-linked, compared to the 20 to 50% that is cross-linked in E. coli
(reviewed by Rubin, Jackson, Brennan and coworkers 1; 2
). The interaction may also be strengthened by the formation of ArfA dimers in vivo, as proposed for Pal 27
. For example, MotB, and to a weaker extent RmpM, crystallize as dimers 22; 23
, and the B domain of ArfA has been proposed to oligomerize 11
. Pal and ArfA are monomeric in solution, both in their free and UMDP-bound states. However, the molecular surface of ArfA C domain displays a distinct electrostatic polarity (electropositive on the side where α3 packs against β3 and negative on the opposite side where α1 packs against α2), and it is tempting to speculate that this charge juxtaposition may assist the assembly of stacked, neighboring C domains on the peptidoglycan polymer as a means of strengthening the cell wall.