Structure of the stationary phase survival protein YuiC from B.subtilis

Protein expression and structure solution

B. subtilis YuiC 32–218 (Uniprot J7JYQ4_BACIU), which lacks only the predicted signal peptide,
was purified after cytosolic expression in E. coli. This protein ran as two peaks on gel filtration corresponding to probable monomer
and dimer fractions based on the elution volume (Additional file 1: Figure S1). The sample also showed partial proteolysis to give a lower molecular
weight band on SDS-PAGE (Additional file 1: Figure S2). Peptide mapping by mass spectrometry indicated that this was likely
to be cleavage at or close to R52 based on the sequence found in the Uniprot database.
NMR spectroscopy of the truncated fragment indicated that there was loss of a series
of sharp peaks in the amide region compared to a full-length sample, consistent with
loss of an unstructured region at the N-terminus (Additional file 1: Figure S3). Subsequent N-terminal truncated constructs (P73-E218 and P73-K217),
designed to remove the disordered regions of the protein, still ran with two peaks
on gel filtration. So far only the first-eluting gel filtration (dimer) peak ever
produced crystals of any construct.

Three structures of YuiC have been solved by molecular replacement based on distant
homology to E. coli MltA (see Experimental Procedures). Two are in space group R3 with two chains forming
a tight dimer in the asymmetric unit. Both structures contain ligands bound to both
chains. One contains the partial substrate N-acetylglucosamine (+NAG) (PDB 4WJT).
The other (+anhydro) was grown in the presence of penta-NAG (PDB 4WLK), but with clear
density in each chain for a 1,6-anhydro-N-acetylglucosamine-1,4-N-acetylglucosamine disaccharide; the expected product of cleavage of a NAG oligosaccharide
substrate. The other structure has no ligand and was solved in space group C222
₁
with a single chain in the asymmetric unit (Apo) (PDB 4WLI). The apo structure forms
a similar dimer to that seen in the R3 structure (in this case with the dimeric symmetry
axis corresponding to the crystallographic two fold axis parallel to the unit cell
b axis). Details of the data collection and refinement are given in Table 1.

Table 1. Data collection and refinement statistics

Fold analysis

The overall fold of each domain of the symmetric dimer consists of a mixed direction
six stranded double psi beta barrel surround by five helices (Fig. 1a). In all the structures, the final two helices and the last strand of the beta barrel
of each half are supplied by the other chain in the dimer. This appears to be a classical
example of crystallographic (sub)domain swapping as defined by Eisenberg 17]. It is likely, and certainly topologically possible, that in the monomeric form seen
in solution the last strand (?6) and final helices (?4 and ?5) of the domain are provided
by the same chain. These three secondary structural elements then swap to form a dimer
at higher protein concentrations (Fig. 1a).

Fig. 1. Structure of YuiC and comparison with MltA. a Dimer of YuiC with NAG bound. Chain A is in magenta and Chain B in blue with the
positions of starts and ends of secondary structure elements labelled. NAG is ball
and stick with carbon in green, oxygen in red and nitrogen in blue. The distance between
the CA of G176 of each chain is shown in Ã…. b Structural Superposition of YuiC structure backbones. +NAG chains in magenta and
blue with ligand in green, +Anhydro chains in red and pale crimson and ligand in dark
purple. Apo chains in green and yellow. c Structural superposition of?+?NAG YuiC in cyan (A72-176) and blue (B177-217) (pseudo
monomer) and MltA from E.coli (PDB 2ae0) 19] in gold. Lower picture is 90° rotation around horizontal of upper. The distance between
A176 and B177 of YuiC is shown in Ã…. d Sequence alignment based on the structural superposition in C with secondary structure
elements labelled, conserved aspartates shown in green and other conserved residues
shown in red. G176, where the domain swap is centred, is coloured yellow and labelled.
Structural superposition used SSM 36] in CCP4MG 37], structural alignment generated by UCSF chimera 38], structures drawn with CCP4MG 37] and alignment with ESPRIPT 39]

The domain swapping is probably enabled by the ability of G176 to adopt the necessary
phi-psi angles to allow either monomer or dimer to form. G176 lies at the end of the
third helix. The CAs of G176 in the two chains are only 4.34 Å apart (Fig. 1a), so movement of a single helix to an “average” position in the monomer is plausible.
Most of the dimer interface interactions would be found in a monomer, the exceptions
are the interface between the two copies of the third helix (residues 166–178). Taking
just the residues 166–178 from the two chains of?+?NAG using PISA 18] 316 Å
²
is buried between the two helices and surrounding linkers with no additional hydrogen
bonds or salt bridges definitely formed in the dimer compared to two separate monomers.
This compares to 3955 Å
²
in the total interface between the A and B chains. Two hydrogen bonds are formed from
residues in this swapping region to other parts of the second chain. E166 side chain
forms a hydrogen bond to T 215 very close to C-terminus of the other chain, and would
presumably maintain this link in the monomer. The hydroxyl of Y172 forms a hydrogen
bond to Glu98 in the other chain. It would take a movement of about 5 Å, rotating
the helix in the right direction to bring G176 to the position of G176 in the other
chain, for this to be an intrachain rather than an interchain hydrogen bond. This
indicates that the dimer should only be slightly more stable than the monomer.

The two ligand bound structures have good electron density for all residues, and superimpose
very well with a RMSD 0.40 Å over 289 CA atoms of the dimer or 0.23 Å over 145 CA
atoms of the pseudo monomer (formed by chain A 72–176 and chain B 177–216) (Fig. 1b). The apo structure is locally less ordered than the substrate bound structures,
lacking density for residues 97–100 and for four residues at the C-terminus compared
to the?+?NAG structure, which just misses one residue at the C-terminus. Otherwise,
the apo form of the pseudo monomer superposes well onto the?+?NAG structure with an
RMSD of 1.15 Å over 135 CA atoms for the pseudo monomer. The domain swapped dimers
superimpose less well with the RMSD for the apo dimer vs?+?NAG of 2.81 Å over 241
CA atoms as a consequence of flexibility in the positioning of the pseudo monomers
within the dimer. The domain swapped dimer corresponds to a two-fold axis parallel
to the third helix which lies at the centre of the dimer interface. In the apo crystals,
the two fold axis is crystallographic.

If one pair of pseudo monomers is superimposed between apo and?+?NAG, it requires
a rotation of 26° and a translation of 6 Å along a screw axis perpendicular to the
third helix to superimpose the second pair of monomers (Fig. 1b) starts at residue 167 just before the third helix, however the two residues with
very large phi/psi angle changes between liganded and apo structures, where most of
the movement arises are G176 (phi/psi?+?NAG ?83/?25 apo ?77/169) and K178 (phi/psi?+?NAG
?139/135 apo ?88/?40). The flexibility of G176 agrees with, but does not prove, our
proposal that a major rearrangement at this residue will generate the non-domain swapped
monomer. The NZ of K178 in the apo structure forms hydrogen bonds to both the main
chain and side chain carbonyls of N173, whereas in the liganded structures this side
chain is pointing in to solvent. Whether this is the cause of the phi/psi angle at
this residue is not clear. The region 176–178 lies away from the sugar binding site,
so the change is not a direct result of ligand binding. However the other end of the
third helix lies quite close to the ligand binding site and the loop that becomes
ordered on ligand binding, so the difference in domain position may be propagated
from sugar binding. However, it is also possible that changes in crystal packing may
be the sole cause of the difference in the position of the second monomer seen in
the apo structure.

Comparison to MltA

MltA (membrane bound lytic transglycosylase A) (PDB 2ae0) 19] is defined as a single domain in SCOP, but as two domains in CATH – (2.40.40.10)
the Barwin-like endoglucanase beta barrel, formed by a section from the N terminus
(residues 20–104) (strands ?1-3) and the C terminal region (243–337) (strands ?10-14),
and an unclassified domain (105–242). MltA superimposes on the YuiC (+NAG) structure
with an RMSD of 2.07 Å over 102 of the 146 modelled residues (72–217) in the pseudo
monomer (Fig. 1c). This is entirely within the beta barrel domain of MltA, which is 180 residues long
and is slightly longer than the ordered region of YuiC (146 residues). Overall YuiC
resembles closely the Barwin-like endoglucanase beta barrel of MltA, but instead of
the second 138 residue domain of MltA, YuiC just has a 10 residue loop linking the
two sections of the barrel. All the beta strands of YuiC have equivalents in MltA
(Fig. 1d). The first strand and adjacent peptide of YuiC (84–96) is overlapped by the second
and third strands of the MltA (82–104), which has an eight residue loop between the
two strands that does not superimpose with YuiC. There is no equivalent of the first
strand and helix of MltA in YuiC. Where the CATH Barwin-like endoglucanase domain
of MltA begins again (243–253), the backbone is very close to YuiC (110–120) in a
small hairpin in both structures. The second to fifth strands of YuiC superimpose
with the tenth to thirteenth of YuiC. Before the final strand of the beta barrel both
have helices, which do not superimpose well. The YuiC helix at this point, ?3, is
where the domain swap begins. The final strand of the beta barrel (?6) is formed by
the last strand of YuiC from the other chain in the domain swap and is equivalent
to the last strand (?14) of MltA. YuiC then has a pair of helices, which are close
in space to the helices at the N-terminus of MltA but are not structurally equivalent.

Ligand binding

Crystallisation of YuiC in the presence of NAG gives a structure with a single well
defined NAG per chain. Incubation of YuiC with 5 mM penta-NAG in the crystallisation
results in two linked sugars in the final structure per chain. One of the sugars is
a NAG that occupies the same ?2 site as the sugar in the crystals grown in the presence
of NAG monomer. The second sugar has clearly formed a 1,6-anhydro reaction product.
The interactions of these compounds with YuiC are shown in Fig. 2a and b and discussed more fully below. Peptidoglycan consists of chains of alternating N-acetylglucosmine
(NAG) and N-acetylmuramic acid (NAM) sugars, cross-linked with peptide chains. The
two sugars differ at the O3 position, where NAM has a lactate, which then links to
the crosslinking peptide, whereas NAG just has an OH. This means that NAM is much
more bulky at the O3 position, which often confers the selectivity in cleavage.

Fig. 2. Interactions with ligands for YuiC and MltA. a Interaction of YuiC (cyan) with NAG (green) (H bonds shown as black dotted lines and non-carbon atoms O red and N blue). b Interaction of YuiC (yellow) with 1,6-anhydrodisaccharide (green). 2Fo-Fc electron density for the ligand at 1.0 sigma shown clipped to 1.5 Å around
the ligand. c Interaction of MltA (magenta) with hexachitose (dark cyan) (PDB 2pi8) 20]. 2pi8 is a D308A mutation to prevent catalysis so D308 (light crimson) from the superposed active MltA (PDB 2ae0) is shown 19]. a-c are superimposed views d superposition of YuiC (yellow) with MltA (magenta) showing the superposition of substrates hexachitose (dark cyan) and the 1,6-anhydrodisaccharide (green). This shows the ligands overlapping at the ?1 and ?2 sites. The sidechains of the
three conserved aspartates giving rise to the 3D domain name are also shown. e The potential clash of the ends of a hexachitose in the YuiC structure showing the
+2 NAG of MltA (2pi8) clashing with chain B domain swapping helix (dark crimson helix and transparent grey surface) and the ?4 NAG clashing with a symmetry related copy of YuiC in the lattice
(dark green and transparent grey surface). Diagrams drawn with CCP4mg 37]

The structure of the catalytically inactive D308A MltA with chitohexose 20] (PDB 2pi8) has six clearly defined NAG sugars, four, ?4 to ?1, on the non-reducing
end before the cleaved bond and two, +1 and +2, at the reducing end. The NAG in?+?NAG
superimposes with the ?2 position in the chitohexose in MltA. The anhydro sugar lies
at the ?1 position and the unmodified NAG lies at the ?2 position in the disaccharide
reaction product. The interaction of the conserved D297 (MltA)/D151 (YuiC) with the
N of the N-acetyl group of the NAG at the ?2 position is conserved (Fig. 2a-c, Table 2). Further hydrogen bonds to this sugar in YuiC are from the non-conserved K102 NZ
to the N-acetyl carbonyl and O3 of NAG. MltA does not have any atoms near K102 NZ
in the superposition and K102 lies in the YuiC insert that replaces a whole domain
in MltA. The OH of S164 in MltA does form an H bond to the ?2 N-acetyl carbonyl, but
lies 4.2 Å from K102NZ in the superposition and does not also interact with O3, and
there is a water (HOH 428) in the YuiC structure at the position of the MltA S164
OH.

Table 2. List of Hydrogen bonds between protein and substrates and conservation in MltA

More generally there is good conservation of the backbone on the D151 side of the
sugar, but little conservation on the other face. The O3 hydroxyl in the ?2 position
forms a hydrogen bond to the backbone carboxyl of L111, which would prevent there
being a N-acetylmuramicacid (NAM) at this position. NAM can easily be accommodated
at the ?1 position as the O3 of the sugar, which has the lactic acid group in NAM
and then the peptide in peptidoglycan, is pointing into the solvent. The ?1 site in
MltA has a hydrogen bond to the main chain carboxyl of residue V298 from the O6 hydroxyl
(Fig. 2c, Table 2). This backbone position is conserved in YuiC, but the hydroxyl has moved away to
form the anhydro product and so this contact is lost in the product. The N of the
N-acetyl group is interacting with the main chain carboxyl of V161 in MltA. This is
roughly equivalent in position to the carbonyl of S99 in YuiC?+?anhydro structure,
which forms a similar interaction, despite the overall fold not being conserved in
this region. The carbonyl oxygen of the acetyl group of the ?1 sugar in the YuiC?+?anhydro
product interacts with two main chain NH groups (S154 and A155), which are conserved
in MltA (G300 and A301), although the carbonyl also interacts with the side chain
OH of S154 in YuiC, which is an extra interaction compared to MltA. In MltA the acetyl
carbonyl is further away and the interaction with G300/A301 is water mediated. Without
a product structure for MltA or an uncleaved substrate in YuiC it is impossible to
determine whether the differences in binding are due to substrate/product differences
or protein differences.

The superposition of the MltA sugars allows us to look more widely at possible sugar
binding sites in YuiC. Intriguingly there is only room for one sugar site on the vacant?+?side
of the cleavage in the?+?NAG and anhydro structure. The +2 sugar in the MltA superposition
clashes with the third (domain swap) helix backbone (Fig. 2e). This would prevent the sugar chain being longer than +1 and so the dimer could
only remove a terminal NAG (ie be an exo glycosidase). However the movement of the
second pseudo monomer of the domain swapped dimer in the apo structure described above,
displaces the third helix away from this position so that this clash is reduced to
ends of side chains, which could adopt other positions. This probably would allow
cleavage within a chain (endo) in the dimer as well, and certainly allow removal of
disaccharides as seen in many lytic transglycosidases. In the pseudo monomer, the
block from the helix probably does not occur and the active site is much more open
so the monomer is likely to be able to cleave in either an endo or exo mode.

The main interactions with the +1 sugar in MltA are formed by residues V161 and Q162,
which lies in the inserted domain in CATH that is not present in YuiC. However, the
hydroxyl of S99 of YuiC, which is part of the much more direct link that replaces
the inserted domain, lies close to the position of the Q162 side chain of MltA and
could potentially hydrogen bond to O3 of the +1 sugar. There is not much space round
the superimposed +1 O3 hydroxyl in YuiC and so the +1 position is likely to be specific
to NAG and not able to house a NAM residue.

Although we can only clearly see two sugars in the product, potentially a third may
be present in the anhydro product in a disordered state. The regions of MltA that
interact with the ?3 and ?4 sugars in MltA are not homologous with YuiC. The superimposed
?4 sugar of MltA collides with a symmetry related molecule of YuiC (Fig. 2e) suggesting that a product with four sugars would not bind in the lattice. Intriguingly
this would be the obvious product of the penta-NAG in the YuiC dimer R3 crystal as
the +2 position is also sterically blocked. It is hard to envisage YuiC having any
positive interaction with a sugar in the ?4 position as the protein does not extend
out that far. Despite being larger, MltA only has limited interaction with the sugar
at the ?4 position. Careful inspection of the ?3 site indicate some waters are in
positions likely to be where hydroxyls of the sugar would be positioned, but if it
is present the sugar is either much more mobile or much less occupied due to cleavage
at a mixture of positions in the penta-NAG. It is more likely that multiple cleavage
events before crystals formed have led to a predominant two sugar anhydro product
and this form bound to the dimer may have been preferentially selected by the lattice.

Catalytic activity

The conserved aspartates of the 3D domain, the Pfam annotation of YuiC (http://pfam.xfam.org/family/3D), superimpose well with the equivalent residues in MltA. YuiC D162 is equivalent
to MltA D308 (A308 in 2pi8); YuiC D129 to MltA D261and YuiC D151 to MltA D297. No
equivalent atoms are further than 1.4 Å apart and all CAs within 0.5 Å.

The conserved D162 is the catalytic carboxylate and is orientated by T91 which is
conserved in MltA (T99) (Fig. 3). A number of proposed mechanisms for MltA have been put forward. In the preferred
mechanism of Van Straaten et al.20] the catalytic aspartate residue is proposed to protonate the leaving hydroxyl at
the +1 position and deprotonate the O6 hydroxyl, which attacks a carbenium ion intermediate
to form the anhydro product. It is proposed in MltA that the carbenium ion is stabilised
by the ?4 helix dipole. This helix lies in the inserted domain which has no equivalent
in YuiC. However in the?+?NAG and anhydro product structures the nearest residues
to the position of the helix in the superposed MltA are E98 and S99. The main chain
carboxyl of S99 is hydrogen bonding to the N of the N-acetyl group of the ?1 anhydrosugar with the side chain of E98 pointing away towards
the side chain of T94. However the stretch of residues from 97 to 100 is disordered
in the apo structure indicating that these residues are flexible and therefore E98
may be able to rearrange and act as a second carboxylate in the reaction mechanism,
either just to stabilise the carbenium ion as the dipole is proposed to do in MltA,
or opening up the possibility of a two carboxylate mechanism analogous to the retaining
lysozymes.

Fig. 3. Schematic of the YuiC active site showing the conservation with MltA. The lower barrel
side shows significant conservation to MltA including the conserved catalytic aspartate
and the residues allowing mechanism 2 of Powell et al.21]. The upper face is not conserved. Substrate assisted catalysis is unlikely because
S154, which is unique to YuiC is holding the acetyl carbonyl in the wrong place for
this mechanism. The helix from MltA (purple) is thought to stabilise the carbenium ion. It is possible that the helix from YuiC
(cyan) may play a similar role or release E98 to act as a second carboxylate, as 97–100
are disordered in the apo structure indicating flexibility in this region. The two
helices are shown in their superimposed positions. Where two numbers are given the
first is B.subtilis YuiC and the second E.coli MltA (2pi8 numbering)

Substrate assisted catalysis has also been proposed for MltA 21]. This would require the ?1 sugar N-acetyl oxygen to be on the opposite face of the
substrate from the O6 that generates the anhydrosugar. However in YuiC the ?1 sugar
N-acetyl group oxygen of the anhydro product is interacting with S154 and is on the
same face as the O6 oxygen would be. Unless the S154 interaction is only formed in
the product then substrate assisted catalysis using the N-acetyl group is unlikely,
as a very large rearrangement of the N-acetyl group is required to place it in position
to assist in substrate catalysis from its position in the product structure. All the
homologous residues for the second mechanism of Powell et al.21] involving Y93 and D151 of YuiC acting in the same way as proposed for Y140 and D393
of N. gonnorrhoeae MltA to abstract the proton from the O6 to promote nucleophilic attack on the carbenium
to form the anhydro product.

Is the monomer or the dimer the true structure?

Zymograms indicate that protein from both peaks of the gel filtration are enzymatically
active and can degrade peptidoglycan after refolding after SDS-PAGE (Fig. 4a-b). However, this does not demonstrate which oligomeric states are active as the refolding
from the unfolded monomer in the gel could have led to either, or a mixture of both,
oligomeric states. A native gel shows no major difference in apparent size or activity
of the samples originating from the monomer and dimer peaks (Fig. 4c-d), probably the very high concentration in the stacking gel has driven the protein
largely into the dimer state. There are some other higher oligomer bands that are
also active, particularly from the monomer peak.

Fig. 4. Zymograms of YuiC. a SDS-PAGE and b denaturing zymogram of YuiC_P73 and YuiC_R52 constructs. Lane 1: YuiC_P73-E218 dimer,
2: YuiC_P73-E218 monomer, 3: YuiC_P73-K217 dimer, 4: YuiC_P73-K217 monomer, M: PageRuler
prestained protein ladder, 5: YuiC_R52-E218 peak 1 (monomer), 6: YuiC_R52-E218 peak
2 (dimer), 7: YuiC_R52-E218 peak 3 (oligomers), 8: YuiC_R52-K217 peak 1 (monomer),
9: YuiC_R52-K217 peak 2 (dimer), 10: YuiC_R52-K217 (oligomers). c Native-PAGE and d native zymogram of YuiC_P73 constructs. Lane 1: Lysozyme, M: NativeMark unstained
protein standard, 2: YuiC_P73-E218 dimer, 3: YuiC_P73-E218 monomer, 4: YuiC_P73-K217
dimer, 5: YuiC_P73-K217 monomer, 6: negative control (Rv3368). Positions of the principal
bands in the native zymogram are marked with red arrows as they are faint

The domain swapped dimer seen in the crystal may be an artefact of high level expression
in the E.coli cytosol and high concentrations used for structural studies, however, we have no
direct evidence for this. Nevertheless, both the monomer and dimer are stable species.
Rerunning samples of either peak, after being frozen for some weeks, gives a single
peak with a similar retention volume on gel filtration as when first run (Additional
file 1: Figure S1). This indicates that both states are stable and there is at best very
slow kinetic interchange between the two.

It is pure speculation as to what the oligomeric state is in B.sutbtilis in vivo. A mixture of the two states is possible, particularly as both are kinetically stable
and probably active. Highly expressing protein in the cytoplasm of E.coli is different from an unknown level of expression of secreted protein in B.subtilis, so the distribution seen in our experiments may not reflect nature. Furthermore
YuiC may interact with the cell wall or other enzymes through the disordered region
at the N-terminus, which could influence its ability to oligomerise. Peptidoglycan
remodelling enzymes are known to interact. RpfB and RpfE bind to RipA and there is
synergy in cleavage seen between the two 22]–24]. Indeed RpfB and RipA assemble into a larger complex with PBP1 at the poles septum
24]. The assembly of multiple peptidoglycan enzymes is a theme also seen in Gram-negative
E. coli25].