BACKGROUND
cft <dig> escherichia coli is an uropathogenic strain responsible for conditions like cystitis and pyelonephritis , severe cases of which may lead to sepsis
<cit> .
uropathogenic e. coli  bacteria are a subclass of expec , a group of pathogens responsible for neonatal meningitis and septicaemia
<cit> .
increasing rates of antimicrobial resistance among uropathogens, complicating the future treatment of such infections, led to the development of vaccine preparations based on specific virulence factors, which unfortunately did not demonstrate long-term protection
<cit> .
hence, a broader approach to vaccine design, including the identification of non-virulence factors through methods such as immunoproteomics and reverse vaccinology  is necessary.
recently, subtractive reverse vaccinology was used to identify a number of antigens present in expec but absent or variable in non-pathogenic strains, suggesting that a broadly protective e. coli vaccine may be possible
<cit> .
the  <dig> kd protein coded by locus c <dig> from cft <dig> e. coli was identified as one of nine potential vaccine candidates against expec and was able to confer protection with an efficacy of 33% in a sepsis mouse model
<cit> .
although an antibody-mediated response is likely to be responsible for the capacity of c <dig> to induce protection in mice, the actual mechanism of action of anti-c <dig> antibodies is still unknown.
recent data from our laboratories have suggested a role for c <dig> in impairing the effector functions of human immunoglobulins indicating that antibodies directed against c <dig> may affect the ability of e. coli to evade the immune system
<cit> .
sequence-based analysis performed with smart
<cit>  and pfam
<cit>  indicates that the protein is composed of sel1-like repeats .
these repeats share a consensus sequence that is responsible for their helix-turn-helix  motif and are named after caenorhabditis elegans sel- <dig> gene product
<cit> .
such motifs are flexible in length, usually comprising 36– <dig> amino-acid residues, with few key positions of small and large hydrophobic residues.
the crystal structure of the helicobacter pylori cysteine-rich protein b
<cit> , considered as a prototype of the structural fold consisting of slr units, reveals the modular architecture with the α/α motifs arrayed in tandems and resulting in a super-helical fold.
structural domains composed of several such motifs are thought to act as interaction scaffolds to mediate protein-protein interactions.
slr units can be present in tandem arrays of up to  <dig> motifs or in groups dispersed throughout the protein sequence.
slr-containing proteins are found in both prokaryotic  and eukaryotic organisms, and are thought to have been acquired by horizontal gene transfer.
unfortunately, only few functional annotations are available for slr proteins.
there is accumulating evidence that c. elegans sel <dig> is involved in degradation of proteins from the endoplasmic reticulum, while the yeast hrd <dig> protein is thought to act as an adaptor protein for membrane-bound complexes and hcpa/b from h. pylori is speculated to be responsible for the adaptation of this bacterium to different hosts.
it could be said that these molecular functions of slr proteins are related, in that they are associated with signal transduction pathways
<cit> .
slr proteins share similar consensus sequence with the much more abundant tpr  protein family, in which tpr units are composed of  <dig> amino-acid residues
<cit> .
the structural topology of tpr-containing proteins was revealed by the structure of the tpr domain of the protein phosphatase  <dig>
<cit> .
it displays a super-helical fold similar to the one characteristic of the slr family.
however, the superposition of this tpr domain with hcpb highlights different super-helix parameters, consequence of different packing angles within and between the repeats.
the region of specific ligand binding, as observed in different tpr domains, is located in the amphipathic groove of the super-helix, with three tandem repeats likely being the optimal minimal length for binding
<cit> .
similar interactions most likely facilitate self-assembly into higher order structures
<cit> .
tpr domains, as mediators of protein-protein interactions, have been implicated in a wide variety of cellular functions, such as transcription, cell cycle, protein translocation, protein degradation and host defence.
due to the non-globular, rather elongated architecture of the repeating tpr and slr units, where stabilization of the fold is achieved mostly through short-range interactions , the energy landscape of these proteins is distinct from that of globular proteins.
inter- and intra-element interactions of such quasi-one-dimensional structures are balanced in such a way that small local perturbations yield large effects, readily facilitating structural transitions that may be related to their biological function
<cit> .
unlike the case of tpr proteins, limited knowledge is available for slr proteins, including fewer available crystal structures as well as functional annotations.
here, we report the  <dig>  Å-resolution crystal structure of c <dig> from cft <dig> e. coli determined by se-met sad phasing.
the structure is composed of  <dig> slr units, which to our knowledge represents the bacterial protein with the highest number of sel1-like repeats solved up to date.
it displays similar packing angles to those found in hcpb/c proteins from h. pylori, however with a distinct mode of overall fold stabilisation.
furthermore, we report the presence of a metal-ion binding site, generally uncharacteristic of tpr and slr proteins.
crystal contacts between the c-terminal tail of c <dig> and the c-terminal section of the amphipathic groove of a molecule belonging to the adjacent asymmetric unit are analysed and their possible biological relevance discussed.
as part of a study of the antigenic properties of c <dig>  the regions of the protein that are recognised by antibodies have been mapped using murine monoclonal iggs.
RESULTS
overall structure
the crystallographic structure of the functional unit  of c <dig> has been solved by sad phasing and refined to a resolution of  <dig>  Å. according to predictions by signalp
<cit> , the first  <dig> amino acids constitute a signal sequence.
the final model has r and rfree values of  <dig> % and  <dig> %, respectively.
the model has acceptable root-mean-square differences for bond lengths and angles  and none of the residues lie in disallowed regions of the ramachandran plot .
one molecule is present in the asymmetric unit of the crystal.
amino-acid numbering in the model sequence reflects that of the functional unit .
adata collected at the id14- <dig> mad beamline of esrf  <cit> .
bnumbers in parentheses refer to the highest resolution shell.
crfree was calculated using a subset  of the reflections not used in the refinement.
c <dig> displays a super-helical fold , containing eleven sel1-like repeats, an n-terminal and two c-terminal helices with probable capping function and a  c-terminal tail.
each repeat consists of two helices, helix- <dig> and helix- <dig>  formed predominantly by thirteen residues, and connected predominantly by a 7-residue loop .
individual repeats, tethered by three-residue loops, stack on top of each other creating an extended super-helical molecule with a continuous hydrophobic core.
this structure can also be viewed as an overlapping array of three-helix bundles.
the right-handed super-helix is approximately 115 Å in length, with a diameter of ~50 Å and a pitch  of 60–65 Å. a complete helical turn comprises about seven to eight slr units.
the n- and c-terminal helices do not have a true slr consensus sequence, but they share structural homology.
a closer inspection of the amino-acid sequence suggests a role in ‘neutralizing’ hydrophobic surfaces on solvent exposed parts of the first and last repeats, hence facilitating the molecule’s solubility.
to date, this is the known structure with the highest number of slr repeats for a bacterial protein.
uncharacteristic of slr and tpr proteins, a metal-ion binding site, occupied by magnesium, was found between repeats  <dig> and  <dig> .
it resides in the negatively charged patch of the amphiphilic concave surface of the super-helix.
finally, the c-terminal region of the molecule contacts the c-terminal tail of the molecule in the adjacent asymmetric unit.
similarities with other slr and tpr proteins
the packing angles of repeats are similar to those observed in the h. pylori cysteine-rich protein c
<cit> , comprised of  <dig> residues  and sharing 47-53% sequence similarity  with c <dig> .
an important difference between the two proteins resides in the inter-repeat disulfide bonds stabilizing the super-helical packing in hcpc, not present in c <dig>  likewise, a shorter homologue , h. pylori cysteine rich protein b
<cit> , has a 39-46% sequence similarity with c <dig> and its repeats are also cross-linked by disulfide bonds.
finally, the putative sel1-repeat protein kpn_ <dig>  from klebsiella pneumoniae ssp.
pneumoniae  shares 45-47% sequence similarity with c <dig>  while hcpc displays a pattern of repeat interactions similar to that found in c <dig>  with slight variation in packing angles within and between the repeats, kpn_ <dig> presents an uncommon packing angle of its fifth repeat that allows contacts with the intra-repeat loops belonging to the second and third repeats.
structure resolution and functional annotation of more slr family proteins shall lead to understanding the necessity for this observed variety.
the first structure of a tpr-motif-containing protein was solved in 1998
<cit>  and, to date, the structure with the largest number of repeats  is that of the tpr domain of o-linked glcnac transferase
<cit> .
a distinct packing angle of the tpr units, as well as within repeats, allows a narrower super-helix in which, unlike slr proteins, convex-face helical contacts are absent and those between concave helices are less extensive.
the significance of these differences in packing between slr and tpr assemblies is yet to be understood, but is likely related to distinguishing target proteins.
structural analysis of inter- and intra-slr interactions
even though the slr family of proteins is known for a low conservation of the consensus sequence of the repeat, c <dig> reproduces this sequence particularly well .
here, the definition of slr derived from that of tpr, as annotated in the smart database has been used.
a repeat is composed of the more tightly packed helices, named helix- <dig> and helix- <dig> .
alternatively, a repeat can be defined as constituted by the two helices packed at a wider angle , as suggested by lüthy and collaborators based on the structures of hcpb and hcpc
<cit> .
the rational for this alternative definition lies in the greater conservation of the latter repeat, reflected mainly in the constant length of the shorter loop between these helices.
herein we retain the smart definition, as one could argue that the repeat should correspond to the entity containing the longer loop, which is an important component in defining the super-helical geometry.
the structure-based sequence alignment in figure
<dig> shows that the majority of the  <dig> repeats retain particularly conserved amino-acid residues at positions  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  indicated in orange, and conserved amino-acid types with dominant representative at positions  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  coloured in blue.
position  <dig>  occupied by aromatic residues in slrs, is interestingly almost exclusively represented by tryptophan throughout the c <dig> repeats.
the slr consensus sequence highlights conserved glycine residues at positions  <dig>   <dig>   <dig> and  <dig>  of which the last three facilitate turns in both intra- and inter-repeat loops while the first one, along with conserved alanine residues at positions  <dig> and  <dig>  allows a close packing of the helices within the repeat.
in turn, alanine residues at positions  <dig> and  <dig> favour the specific packing angle between repeats .
slr proteins, unlike their tpr analogues, have few contacts across helix- <dig> units .
in c <dig>  they are mostly facilitated by the conserved tryptophan at position  <dig> , characteristic for this protein .
interestingly, c <dig> helix- <dig> units have a slight kink, not found in hcpc, which effectively reduces the distance between these units at the side of the inter-repeat loop hence allowing the key positioning of w28r .
on the other hand, existing interactions between helix- <dig> units  tend to be less conserved .
the angular geometry between repeats is mainly dictated by an inter-repeat hydrophobic core centred at conserved residue l7r in helix-1′ .
l7r is in almost all cases in close contact with w28r in helix-2′, with residues 26r , 29r , 30r  and a33r in helix- <dig> and with y11r in the same helix-1′.
further contacts between helix- <dig> and helix-1′ include hydrophobic interactions between residues 10r and 26r, the interaction between residues 6r  and 29r and those between the conserved y11r and 30r, with the frequent presence of a hydrogen bond between the respective hydroxyl and guanidinium groups.
also, 30r  is often hydrogen bonded via structural water to 24r   .
in the convex face, the inter-repeat hydrophobic core is protected from solvent by residues 26r and 30r from helix- <dig>  y11r from helix-1′, v18r from the intra-repeat loop and w28r from helix-2′.
other conserved residue types  play an important role in intra-repeat interactions, such as hydrogen-bonded glutamine residues at positions  <dig> of helix- <dig> and  <dig> of helix- <dig>  the stacking of y11r and 24r , along with the already mentioned l7r and w28r contact .
intra-repeat loops mainly consist of seven residues, with slr <dig> containing one additional residue and slr <dig> four residues less.
such relatively long loops are important for sufficient inter-repeat packing at wide angles, i.e.
the length of these loops governs to a certain extent the stability of the inter-repeat geometry by capping the inter-repeat hydrophobic core.
loop tethering in the conformation observed in the structure is achieved mainly by interactions of conserved v18r with the inter-repeat hydrophobic core .
the inter-repeat loop is shorter, three-residue long, and allows anti-parallel helical packing as well to limit the angle of inter-repeat helices to a certain degree .
compared to its closest homologue hcpc, c <dig> possesses greater sequence conservation and a different means for slr-fold stabilisation.
the hcp family is unique among slr proteins in that, in addition to the contributions from the constituent hydrophobic inter-repeat patch and the intra-repeat loop, fold stabilisation is achieved by disulfide bond tethering the c-terminal end of one repeat and the n-terminal end of the next repeat
<cit> .
in the case of c <dig>  which displays a very similar inter-repeat angle, the disulfide-bond effect is most likely substituted by w28r-mediated interactions.
metal-ion binding site between slrs  <dig> and 4
electron density corresponding to a magnesium ion is identified in the c <dig> structure, with the metal-ion-binding site located between repeats slr <dig> and slr <dig>  h <dig> in slr <dig>  e <dig> in slr <dig> and its intra-repeat-loop residue d <dig>  along with three water molecules, constitute the octahedral coordination ligands of the magnesium ion .
sequence alignment of slr units does not reveal any other potential metal-ion-binding sites, of similar composition, in the protein.
interestingly, the hydrophobic inter-repeat core is not as efficiently packed between repeats  <dig> and  <dig>  this appears mainly due to alanine in position  <dig> of slr <dig> , occupied by larger residues in other repeats, and the corresponding loss of the interaction with positions  <dig> and  <dig> of slr <dig> .
castp, an application for the detection of cavities in proteins
<cit> , identifies a pocket between slr <dig> and  <dig>  as one of the top two in the slr regions, where the other identified pocket is located between slrs  <dig> and  <dig> that lacks the intra-repeat loop.
in the case of slrs 3– <dig>  fewer important interactions between helix-1′ and helices  <dig> and  <dig> along with somewhat weaker intra-repeat loop tethering, in the absence of other compensatory mechanisms, would decrease the stability of the inter-repeat packing.
mg2+ coordination by residues of the intra-repeat loop, helix- <dig> and helix-1′ likely represents a means of stability/flexibility regulation in this region, for a currently unknown purpose.
the significance of the intra-repeat-loop absence between slrs  <dig> and  <dig>  which could lead to a certain degree of instability in this region , is also unknown.
as the likely origin of the magnesium ion in the c <dig> structure is mgcl <dig> used in the crystallization conditions, the natural metal ion for this system and its binding specificities remain to be determined.
real-time quantitative pcr revealed that c <dig> mrna levels in the uropathogenic strain cft <dig> grown in luria-bertani  medium were higher in the presence of specific ion chelators , suggesting a scenario where ions that are ligands to the protein are also regulators of its expression .
unconventional iron binding, like the triad his-glu-asp involved in magnesium-ion coordination in the crystal structure of c <dig>  is found in x-ray structures of proteins closely related to ferritin and dna-binding proteins from starved cells , e.g.
dps from mycobacterium smegmatis  or antigen tpf <dig> from treponema pallidum .
interestingly, all these species are dodecameric entities presenting a spherical shell with a large inner cavity.
the iron ion binds inside the cavity at the interface between two adjacent monomers.
the histidine on one side, and glutamate and aspartate on the other, are provided by different subunits and configure the metal-binding site .
in all dps-like structures, the iron ion presents a possible tetrahedral or trigonal bipyramid geometry, given that water molecules involved in coordination might not be observed due to low resolution .
it cannot be excluded that the coordination of the metal-ion in c <dig> could be of such lower order in a potentially native iron-bound structure.
metal-ion binding, as evident from the pdb repository, is not common in slr and tpr-containing proteins.
however, an example where metal-ion binding might be partly responsible for dynamics and ligand-binding regulation is that of human pex5p receptor.
sr2+ binding  in the protein's tpr domain hinge region, even though in a coordination not resembling the case of c <dig>  leads to near rigid-body movement of its two halves  and less overall conformational flexibility of the domain
<cit> .
metal ions play a role in many important functions in proteins, including stability, conformational changes, folding and assembly.
one can speculate that for c <dig> the stabilisation of the slr 3– <dig> region could represent a means of regulation of overall conformational flexibility, and in turn affect ligand  binding .
clearly, further investigation of metal-ion binding, its specificity and functional role will be required in order to assess these hypotheses.
crystal contacts between the c-terminal super-helical groove of one molecule and the c-terminal tail of the molecule belonging to the adjacent asymmetric unit
in the crystal packing the concave surface of the super-helix in the region of repeats  <dig>   <dig> and  <dig> of one molecule interacts with the c-terminal tail of a symmetry-related molecule .
interestingly, similar c-terminal tail interactions have already been observed in other slr proteins, such as hcpc .
interactions are predominantly of polar character accompanied by van der waals contacts.
n <dig>  belonging to repeat  <dig>  participates in bidentate hydrogen-bonding interactions with the backbone carbonyl oxygen and nitrogen atoms of the c-terminal tail's q <dig>  n <dig> of repeat  <dig> is near the c-terminal tail's a <dig> and could be engaged in similar interactions with its backbone atoms, upon rotameric change .
these interactions can be categorised as anchoring, non-specific for peptide binding in such type of groove.
specificity might be governed by non-polar interactions of the tail peptide with the inter-repeat core and polar interactions with the solvent exposed repeat area.
thus, residues responsible for shape complementarity comprise a <dig> and a <dig> of the c-terminal tail in van der waals contact with a <dig> and a <dig>  a <dig>  t <dig> of the second molecule, respectively.
polar interactions involve s <dig> hydrogen bonding to r <dig> in slr <dig> and k <dig> in slr <dig>  while d <dig> and t <dig> in slr <dig> take part in charge-charge and hydrogen-bond interactions with k <dig> in the c-terminal tail, stacking against y <dig> of slr <dig>  additional stacking interactions are observed between f <dig> belonging to one of the c-terminal helices and k <dig> at the c-terminal end .
the last three residues of the c-terminus, for which lower electron density is observed, do not really contribute to the interactions within this concave pocket.
some similarity is shared with the mode of target-protein/peptide binding of tpr proteins, such as receptor for peroxisomal uptake-pex <dig>  hsp70/hsp <dig> organizing protein-hop , fkbp <dig> and pp <dig>  in these, tpr tandems recognise the c-terminal eevd signal sequence of the target protein/peptide by a “carboxylate clamp”
<cit>  and likely also utilizing bidentate hydrogen-bonding interactions between conserved asparagine residues lining the super-helical groove and target peptide backbone atoms
<cit> .
figure
<dig> highlights this type of hydrogen bonding, involving a conserved asparagine residue, in c <dig>  hcpc and hop/hsp <dig>  in most cases, the binding pocket establishing primary interactions through the target’s c-terminal tail is composed of three tpr repeats, in line with three tandem tpr domains being the most populated, suggesting that these represent the minimal functional binding unit.
secondary interactions are often important for establishing specificity and are thought to lie outside of this primary region, as shown for hop
<cit> .
cortajarena et al.
also emphasised the importance of both short-range interactions and long-range electrostatics as determinants of specificity
<cit> .
on the other hand, there are examples of self association for tpr proteins, such as dimerisation of the sgt <dig> plant protein
<cit>  or oligomerisation of the mama bacterial protein , that involve their terminal helices binding in the super-helical groove regions
<cit> .
for a number of tpr-containing proteins it has been shown that self-association can serve to regulate their biological function.
se-hplc analysis of c <dig> shows that aggregates/oligomers are present in very little amount , although an exhaustive study of c <dig> oligomerisation has not been performed.
the potential biological significance of the observed crystal contact in c <dig> is revealed by comparison with the known tpr ligand-binding examples.
indeed, they share common features like binding in the concave area of the super-helix with similar peptide-backbone anchoring mode and three-tandem slr domains as binding pocket.
correlation between the crystal contacts observed in the tpr protein cyp <dig> and its interactions with the natural ligand  in solution
<cit>  further suggests the possibility of a similar scenario in the c <dig> case, with yet unidentified target protein/peptide .
epitope mapping
mapping of c <dig> epitopes for murine monoclonal antibodies was performed as part of the general characterization of this protein as a potential vaccine candidate, alongside presenting an opportunity to further investigate binding regions in c <dig>  proteolytic digestion of the antigen following its incubation with monoclonal antibodies  did not result in identification of any epitope-containing peptides.
however, epitope-containing peptides were captured from partial digestion of c <dig>  with gluc  or lysc  .
the sequences of the epitope-containing peptides map to helix- <dig> of slr <dig> for mab16h8/g <dig>  helix- <dig> of slr <dig> for mab17a7-c <dig> and helix- <dig> of slr <dig> and helix- <dig> of slr <dig> for mab14e7/d <dig> .
failure to immunocapture the products of lysc or trypsin cleavage  by mab17a7-c <dig> and mab14e7/d <dig> further narrows down the important epitope components to the intra-repeat loop of slr <dig>  for mab17a7-c <dig> and to the outer helix- <dig> of slr <dig>  for mab14e7/d <dig>  respectively.
these are in agreement with no steric hindrance to the access and binding of the antibody to these convex areas of the super-helix, and partly overlap with regions that are involved in mg2+ binding  or belong to the c-terminal-tail binding groove .
how relevant this observation is with respect to the previously discussed roles of these regions remains to be further investigated.
CONCLUSIONS
we have solved the structure of c <dig> from uropathogenic escherichia coli to  <dig>  Å resolution.
this antigen displays a super-helical sel1-like repeat fold with eleven slr units and a remarkably preserved consensus repeat sequence.
it shares high structural similarity with its closest homologue of known three-dimensional structure, hcpc from helicobacter pylori, albeit with differences in how the slr-fold is stabilized.
while disulfide bridges in hcpc lock the characteristic inter-repeat geometry, in c <dig> a conserved tryptophan residue at repeat position  <dig> appears to contribute fundamentally in maintaining the same geometry by strengthening the inter-repeat hydrophobic core.
metal ion binding, generally uncharacteristic of slr proteins, is observed between slr units  <dig> and  <dig>  suggesting a regulatory role in conformational flexibility.
furthermore, crystal contacts observed between molecules belonging to neighbour asymmetric units share similarity to contacts characteristic for tpr-protein interactions with their physiological targets, suggesting a potential physiological interaction mode of c <dig> with yet unidentified targets.
the structure of c <dig> is a first step for its functional characterisation and opens the door to the possibility of redesigning this antigen for vaccine-development purposes.

 cft escherichia coli is an uropathogenic strain responsible for conditions like cystitis and pyelonephritis, severe cases of which may lead to sepsis. uropathogenic e. coli bacteria are a subclass of expec, a group of pathogens responsible for neonatal meningitis and septicaemia. increasing rates of antimicrobial resistance among uropathogens, complicating the future treatment of such infections, led to the development of vaccine preparations based on specific virulence factors, which unfortunately did not demonstrate long-term protection. hence, a broader approach to vaccine design, including the identification of non-virulence factors through methods such as immunoproteomics and reverse vaccinology is necessary. recently, subtractive reverse vaccinology was used to identify a number of antigens present in expec but absent or variable in non-pathogenic strains, suggesting that a broadly protective e. coli vaccine may be possible. the kd protein coded by locus c from cft e. coli was identified as one of nine potential vaccine candidates against expec and was able to confer protection with an efficacy of 33% in a sepsis mouse model. although an antibody-mediated response is likely to be responsible for the capacity of c to induce protection in mice, the actual mechanism of action of anti-c antibodies is still unknown. recent data from our laboratories have suggested a role for c in impairing the effector functions of human immunoglobulins indicating that antibodies directed against c may affect the ability of e. coli to evade the immune system. sequence-based analysis performed with smart and pfam indicates that the protein is composed of sel1-like repeats. these repeats share a consensus sequence that is responsible for their helix-turn-helix motif and are named after caenorhabditis elegans sel- gene product. such motifs are flexible in length, usually comprising 36– amino-acid residues, with few key positions of small and large hydrophobic residues. the crystal structure of the helicobacter pylori cysteine-rich protein b, considered as a prototype of the structural fold consisting of slr units, reveals the modular architecture with the α/α motifs arrayed in tandems and resulting in a super-helical fold. structural domains composed of several such motifs are thought to act as interaction scaffolds to mediate protein-protein interactions. slr units can be present in tandem arrays of up to motifs or in groups dispersed throughout the protein sequence. slr-containing proteins are found in both prokaryotic and eukaryotic organisms, and are thought to have been acquired by horizontal gene transfer. unfortunately, only few functional annotations are available for slr proteins. there is accumulating evidence that c. elegans sel is involved in degradation of proteins from the endoplasmic reticulum, while the yeast hrd protein is thought to act as an adaptor protein for membrane-bound complexes and hcpa/b from h. pylori is speculated to be responsible for the adaptation of this bacterium to different hosts. it could be said that these molecular functions of slr proteins are related, in that they are associated with signal transduction pathways. slr proteins share similar consensus sequence with the much more abundant tpr protein family, in which tpr units are composed of amino-acid residues. the structural topology of tpr-containing proteins was revealed by the structure of the tpr domain of the protein phosphatase. it displays a super-helical fold similar to the one characteristic of the slr family. however, the superposition of this tpr domain with hcpb highlights different super-helix parameters, consequence of different packing angles within and between the repeats. the region of specific ligand binding, as observed in different tpr domains, is located in the amphipathic groove of the super-helix, with three tandem repeats likely being the optimal minimal length for binding. similar interactions most likely facilitate self-assembly into higher order structures. tpr domains, as mediators of protein-protein interactions, have been implicated in a wide variety of cellular functions, such as transcription, cell cycle, protein translocation, protein degradation and host defence. due to the non-globular, rather elongated architecture of the repeating tpr and slr units, where stabilization of the fold is achieved mostly through short-range interactions, the energy landscape of these proteins is distinct from that of globular proteins. inter- and intra-element interactions of such quasi-one-dimensional structures are balanced in such a way that small local perturbations yield large effects, readily facilitating structural transitions that may be related to their biological function. unlike the case of tpr proteins, limited knowledge is available for slr proteins, including fewer available crystal structures as well as functional annotations. here, we report the  Å-resolution crystal structure of c from cft e. coli determined by se-met sad phasing. the structure is composed of slr units, which to our knowledge represents the bacterial protein with the highest number of sel1-like repeats solved up to date. it displays similar packing angles to those found in hcpb/c proteins from h. pylori, however with a distinct mode of overall fold stabilisation. furthermore, we report the presence of a metal-ion binding site, generally uncharacteristic of tpr and slr proteins. crystal contacts between the c-terminal tail of c and the c-terminal section of the amphipathic groove of a molecule belonging to the adjacent asymmetric unit are analysed and their possible biological relevance discussed. as part of a study of the antigenic properties of c the regions of the protein that are recognised by antibodies have been mapped using murine monoclonal iggs.  overall structure the crystallographic structure of the functional unit of c has been solved by sad phasing and refined to a resolution of  Å. according to predictions by signalp, the first amino acids constitute a signal sequence. the final model has r and rfree values of % and %, respectively. the model has acceptable root-mean-square differences for bond lengths and angles and none of the residues lie in disallowed regions of the ramachandran plot. one molecule is present in the asymmetric unit of the crystal. amino-acid numbering in the model sequence reflects that of the functional unit. adata collected at the id14- mad beamline of esrf. bnumbers in parentheses refer to the highest resolution shell. crfree was calculated using a subset of the reflections not used in the refinement. c displays a super-helical fold, containing eleven sel1-like repeats, an n-terminal and two c-terminal helices with probable capping function and a c-terminal tail. each repeat consists of two helices, helix- and helix- formed predominantly by thirteen residues, and connected predominantly by a 7-residue loop. individual repeats, tethered by three-residue loops, stack on top of each other creating an extended super-helical molecule with a continuous hydrophobic core. this structure can also be viewed as an overlapping array of three-helix bundles. the right-handed super-helix is approximately 115 Å in length, with a diameter of ~50 Å and a pitch of 60–65 Å. a complete helical turn comprises about seven to eight slr units. the n- and c-terminal helices do not have a true slr consensus sequence, but they share structural homology. a closer inspection of the amino-acid sequence suggests a role in ‘neutralizing’ hydrophobic surfaces on solvent exposed parts of the first and last repeats, hence facilitating the molecule’s solubility. to date, this is the known structure with the highest number of slr repeats for a bacterial protein. uncharacteristic of slr and tpr proteins, a metal-ion binding site, occupied by magnesium, was found between repeats and. it resides in the negatively charged patch of the amphiphilic concave surface of the super-helix. finally, the c-terminal region of the molecule contacts the c-terminal tail of the molecule in the adjacent asymmetric unit. similarities with other slr and tpr proteins the packing angles of repeats are similar to those observed in the h. pylori cysteine-rich protein c, comprised of residues and sharing 47-53% sequence similarity with c. an important difference between the two proteins resides in the inter-repeat disulfide bonds stabilizing the super-helical packing in hcpc, not present in c likewise, a shorter homologue, h. pylori cysteine rich protein b, has a 39-46% sequence similarity with c and its repeats are also cross-linked by disulfide bonds. finally, the putative sel1-repeat protein kpn_ from klebsiella pneumoniae ssp. pneumoniae shares 45-47% sequence similarity with c while hcpc displays a pattern of repeat interactions similar to that found in c with slight variation in packing angles within and between the repeats, kpn_ presents an uncommon packing angle of its fifth repeat that allows contacts with the intra-repeat loops belonging to the second and third repeats. structure resolution and functional annotation of more slr family proteins shall lead to understanding the necessity for this observed variety. the first structure of a tpr-motif-containing protein was solved in 1998 and, to date, the structure with the largest number of repeats is that of the tpr domain of o-linked glcnac transferase. a distinct packing angle of the tpr units, as well as within repeats, allows a narrower super-helix in which, unlike slr proteins, convex-face helical contacts are absent and those between concave helices are less extensive. the significance of these differences in packing between slr and tpr assemblies is yet to be understood, but is likely related to distinguishing target proteins. structural analysis of inter- and intra-slr interactions even though the slr family of proteins is known for a low conservation of the consensus sequence of the repeat, c reproduces this sequence particularly well. here, the definition of slr derived from that of tpr, as annotated in the smart database has been used. a repeat is composed of the more tightly packed helices, named helix- and helix-. alternatively, a repeat can be defined as constituted by the two helices packed at a wider angle, as suggested by lüthy and collaborators based on the structures of hcpb and hcpc. the rational for this alternative definition lies in the greater conservation of the latter repeat, reflected mainly in the constant length of the shorter loop between these helices. herein we retain the smart definition, as one could argue that the repeat should correspond to the entity containing the longer loop, which is an important component in defining the super-helical geometry. the structure-based sequence alignment in figure  shows that the majority of the repeats retain particularly conserved amino-acid residues at positions and indicated in orange, and conserved amino-acid types with dominant representative at positions and coloured in blue. position occupied by aromatic residues in slrs, is interestingly almost exclusively represented by tryptophan throughout the c repeats. the slr consensus sequence highlights conserved glycine residues at positions and of which the last three facilitate turns in both intra- and inter-repeat loops while the first one, along with conserved alanine residues at positions and allows a close packing of the helices within the repeat. in turn, alanine residues at positions and favour the specific packing angle between repeats. slr proteins, unlike their tpr analogues, have few contacts across helix- units. in c they are mostly facilitated by the conserved tryptophan at position, characteristic for this protein. interestingly, c helix- units have a slight kink, not found in hcpc, which effectively reduces the distance between these units at the side of the inter-repeat loop hence allowing the key positioning of w28r. on the other hand, existing interactions between helix- units tend to be less conserved. the angular geometry between repeats is mainly dictated by an inter-repeat hydrophobic core centred at conserved residue l7r in helix-1′. l7r is in almost all cases in close contact with w28r in helix-2′, with residues 26r, 29r, 30r and a33r in helix- and with y11r in the same helix-1′. further contacts between helix- and helix-1′ include hydrophobic interactions between residues 10r and 26r, the interaction between residues 6r and 29r and those between the conserved y11r and 30r, with the frequent presence of a hydrogen bond between the respective hydroxyl and guanidinium groups. also, 30r is often hydrogen bonded via structural water to 24r. in the convex face, the inter-repeat hydrophobic core is protected from solvent by residues 26r and 30r from helix- y11r from helix-1′, v18r from the intra-repeat loop and w28r from helix-2′. other conserved residue types play an important role in intra-repeat interactions, such as hydrogen-bonded glutamine residues at positions of helix- and of helix- the stacking of y11r and 24r, along with the already mentioned l7r and w28r contact. intra-repeat loops mainly consist of seven residues, with slr containing one additional residue and slr four residues less. such relatively long loops are important for sufficient inter-repeat packing at wide angles, i.e. the length of these loops governs to a certain extent the stability of the inter-repeat geometry by capping the inter-repeat hydrophobic core. loop tethering in the conformation observed in the structure is achieved mainly by interactions of conserved v18r with the inter-repeat hydrophobic core. the inter-repeat loop is shorter, three-residue long, and allows anti-parallel helical packing as well to limit the angle of inter-repeat helices to a certain degree. compared to its closest homologue hcpc, c possesses greater sequence conservation and a different means for slr-fold stabilisation. the hcp family is unique among slr proteins in that, in addition to the contributions from the constituent hydrophobic inter-repeat patch and the intra-repeat loop, fold stabilisation is achieved by disulfide bond tethering the c-terminal end of one repeat and the n-terminal end of the next repeat. in the case of c which displays a very similar inter-repeat angle, the disulfide-bond effect is most likely substituted by w28r-mediated interactions. metal-ion binding site between slrs and 4 electron density corresponding to a magnesium ion is identified in the c structure, with the metal-ion-binding site located between repeats slr and slr h in slr e in slr and its intra-repeat-loop residue d along with three water molecules, constitute the octahedral coordination ligands of the magnesium ion. sequence alignment of slr units does not reveal any other potential metal-ion-binding sites, of similar composition, in the protein. interestingly, the hydrophobic inter-repeat core is not as efficiently packed between repeats and this appears mainly due to alanine in position of slr, occupied by larger residues in other repeats, and the corresponding loss of the interaction with positions and of slr. castp, an application for the detection of cavities in proteins, identifies a pocket between slr and as one of the top two in the slr regions, where the other identified pocket is located between slrs and that lacks the intra-repeat loop. in the case of slrs 3– fewer important interactions between helix-1′ and helices and along with somewhat weaker intra-repeat loop tethering, in the absence of other compensatory mechanisms, would decrease the stability of the inter-repeat packing. mg2+ coordination by residues of the intra-repeat loop, helix- and helix-1′ likely represents a means of stability/flexibility regulation in this region, for a currently unknown purpose. the significance of the intra-repeat-loop absence between slrs and which could lead to a certain degree of instability in this region, is also unknown. as the likely origin of the magnesium ion in the c structure is mgcl used in the crystallization conditions, the natural metal ion for this system and its binding specificities remain to be determined. real-time quantitative pcr revealed that c mrna levels in the uropathogenic strain cft grown in luria-bertani medium were higher in the presence of specific ion chelators, suggesting a scenario where ions that are ligands to the protein are also regulators of its expression. unconventional iron binding, like the triad his-glu-asp involved in magnesium-ion coordination in the crystal structure of c is found in x-ray structures of proteins closely related to ferritin and dna-binding proteins from starved cells, e.g. dps from mycobacterium smegmatis or antigen tpf from treponema pallidum. interestingly, all these species are dodecameric entities presenting a spherical shell with a large inner cavity. the iron ion binds inside the cavity at the interface between two adjacent monomers. the histidine on one side, and glutamate and aspartate on the other, are provided by different subunits and configure the metal-binding site. in all dps-like structures, the iron ion presents a possible tetrahedral or trigonal bipyramid geometry, given that water molecules involved in coordination might not be observed due to low resolution. it cannot be excluded that the coordination of the metal-ion in c could be of such lower order in a potentially native iron-bound structure. metal-ion binding, as evident from the pdb repository, is not common in slr and tpr-containing proteins. however, an example where metal-ion binding might be partly responsible for dynamics and ligand-binding regulation is that of human pex5p receptor. sr2+ binding in the protein's tpr domain hinge region, even though in a coordination not resembling the case of c leads to near rigid-body movement of its two halves and less overall conformational flexibility of the domain. metal ions play a role in many important functions in proteins, including stability, conformational changes, folding and assembly. one can speculate that for c the stabilisation of the slr 3– region could represent a means of regulation of overall conformational flexibility, and in turn affect ligand binding. clearly, further investigation of metal-ion binding, its specificity and functional role will be required in order to assess these hypotheses. crystal contacts between the c-terminal super-helical groove of one molecule and the c-terminal tail of the molecule belonging to the adjacent asymmetric unit in the crystal packing the concave surface of the super-helix in the region of repeats and of one molecule interacts with the c-terminal tail of a symmetry-related molecule. interestingly, similar c-terminal tail interactions have already been observed in other slr proteins, such as hcpc. interactions are predominantly of polar character accompanied by van der waals contacts. n belonging to repeat participates in bidentate hydrogen-bonding interactions with the backbone carbonyl oxygen and nitrogen atoms of the c-terminal tail's q n of repeat is near the c-terminal tail's a and could be engaged in similar interactions with its backbone atoms, upon rotameric change. these interactions can be categorised as anchoring, non-specific for peptide binding in such type of groove. specificity might be governed by non-polar interactions of the tail peptide with the inter-repeat core and polar interactions with the solvent exposed repeat area. thus, residues responsible for shape complementarity comprise a and a of the c-terminal tail in van der waals contact with a and a a t of the second molecule, respectively. polar interactions involve s hydrogen bonding to r in slr and k in slr while d and t in slr take part in charge-charge and hydrogen-bond interactions with k in the c-terminal tail, stacking against y of slr additional stacking interactions are observed between f belonging to one of the c-terminal helices and k at the c-terminal end. the last three residues of the c-terminus, for which lower electron density is observed, do not really contribute to the interactions within this concave pocket. some similarity is shared with the mode of target-protein/peptide binding of tpr proteins, such as receptor for peroxisomal uptake-pex hsp70/hsp organizing protein-hop, fkbp and pp in these, tpr tandems recognise the c-terminal eevd signal sequence of the target protein/peptide by a “carboxylate clamp” and likely also utilizing bidentate hydrogen-bonding interactions between conserved asparagine residues lining the super-helical groove and target peptide backbone atoms. figure  highlights this type of hydrogen bonding, involving a conserved asparagine residue, in c hcpc and hop/hsp in most cases, the binding pocket establishing primary interactions through the target’s c-terminal tail is composed of three tpr repeats, in line with three tandem tpr domains being the most populated, suggesting that these represent the minimal functional binding unit. secondary interactions are often important for establishing specificity and are thought to lie outside of this primary region, as shown for hop. cortajarena et al. also emphasised the importance of both short-range interactions and long-range electrostatics as determinants of specificity. on the other hand, there are examples of self association for tpr proteins, such as dimerisation of the sgt plant protein or oligomerisation of the mama bacterial protein, that involve their terminal helices binding in the super-helical groove regions. for a number of tpr-containing proteins it has been shown that self-association can serve to regulate their biological function. se-hplc analysis of c shows that aggregates/oligomers are present in very little amount, although an exhaustive study of c oligomerisation has not been performed. the potential biological significance of the observed crystal contact in c is revealed by comparison with the known tpr ligand-binding examples. indeed, they share common features like binding in the concave area of the super-helix with similar peptide-backbone anchoring mode and three-tandem slr domains as binding pocket. correlation between the crystal contacts observed in the tpr protein cyp and its interactions with the natural ligand in solution further suggests the possibility of a similar scenario in the c case, with yet unidentified target protein/peptide. epitope mapping mapping of c epitopes for murine monoclonal antibodies was performed as part of the general characterization of this protein as a potential vaccine candidate, alongside presenting an opportunity to further investigate binding regions in c proteolytic digestion of the antigen following its incubation with monoclonal antibodies did not result in identification of any epitope-containing peptides. however, epitope-containing peptides were captured from partial digestion of c with gluc or lysc. the sequences of the epitope-containing peptides map to helix- of slr for mab16h8/g helix- of slr for mab17a7-c and helix- of slr and helix- of slr for mab14e7/d. failure to immunocapture the products of lysc or trypsin cleavage by mab17a7-c and mab14e7/d further narrows down the important epitope components to the intra-repeat loop of slr for mab17a7-c and to the outer helix- of slr for mab14e7/d respectively. these are in agreement with no steric hindrance to the access and binding of the antibody to these convex areas of the super-helix, and partly overlap with regions that are involved in mg2+ binding or belong to the c-terminal-tail binding groove. how relevant this observation is with respect to the previously discussed roles of these regions remains to be further investigated.  we have solved the structure of c from uropathogenic escherichia coli to  Å resolution. this antigen displays a super-helical sel1-like repeat fold with eleven slr units and a remarkably preserved consensus repeat sequence. it shares high structural similarity with its closest homologue of known three-dimensional structure, hcpc from helicobacter pylori, albeit with differences in how the slr-fold is stabilized. while disulfide bridges in hcpc lock the characteristic inter-repeat geometry, in c a conserved tryptophan residue at repeat position appears to contribute fundamentally in maintaining the same geometry by strengthening the inter-repeat hydrophobic core. metal ion binding, generally uncharacteristic of slr proteins, is observed between slr units and suggesting a regulatory role in conformational flexibility. furthermore, crystal contacts observed between molecules belonging to neighbour asymmetric units share similarity to contacts characteristic for tpr-protein interactions with their physiological targets, suggesting a potential physiological interaction mode of c with yet unidentified targets. the structure of c is a first step for its functional characterisation and opens the door to the possibility of redesigning this antigen for vaccine-development purposes. 
crystal contacts are observed between the c-terminal tail of one molecule and the c-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats. the location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. the protein coded by locus c from cft e. coli was identified as one of nine potential vaccine candidates against expec and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. the stabilising effect of disulfide bridges in hcpc is replaced in c by a strengthening of the inter-repeat hydrophobic core. these findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen. a metal-ion binding site, uncharacteristic of slr proteins, is detected between slr units and in the region of the inter-repeat hydrophobic core. c lacks functional annotation and structurally belongs to the sel1-like repeat family. the structure is composed of slr units in a topological organisation that highly resembles that found in hcpc from helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. c5321sel1-like repeatcrystal structuresuper-helical foldantigenuropathogenic escherichia coli increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic e. coli strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective escherichia coli vaccine. herein, as part of the general characterization of this potential antigen, we have focused on its structural properties.
 increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic e. coli strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective escherichia coli vaccine. the protein coded by locus c from cft e. coli was identified as one of nine potential vaccine candidates against expec and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c lacks functional annotation and structurally belongs to the sel1-like repeat family. herein, as part of the general characterization of this potential antigen, we have focused on its structural properties.  we report the  Å-resolution crystal structure of c from cft e. coli determined by se-met sad phasing. the structure is composed of slr units in a topological organisation that highly resembles that found in hcpc from helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. the stabilising effect of disulfide bridges in hcpc is replaced in c by a strengthening of the inter-repeat hydrophobic core. a metal-ion binding site, uncharacteristic of slr proteins, is detected between slr units and in the region of the inter-repeat hydrophobic core. crystal contacts are observed between the c-terminal tail of one molecule and the c-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats.  the structure of antigen c presents a mode of stabilization of the slr fold different from that observed in close homologs of known structure. the location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. these findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen. c5321sel1-like repeatcrystal structuresuper-helical foldantigenuropathogenic escherichia coli 