The crystal structure of NlpI A prokaryotic tetratricopeptide repeat protein with a globular fold Christopher G M Wilson1, Tommi Kajander1 and Lynne Regan1,2 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Department of Chemistry, Yale University, New Haven, CT, USA Keywords crystal structure; NlpI; lipoprotein; tetratricopeptide; TPR Correspondence L Regan, Yale University, PO Box 208114, New Haven, CT 06520–8114, USA Fax: +1 203432 5767 Tel: +1 203432 5566 E-mail: lynne.regan@yale.edu (Received September 2004, revised 19 September 2004, accepted 19 September 2004) doi:10.1111/j.1432-1033.2004.04397.x There are several different families of repeat proteins In each, a distinct structural motif is repeated in tandem to generate an elongated structure The nonglobular, extended structures that result are particularly well suited to present a large surface area and to function as interaction domains Many repeat proteins have been demonstrated experimentally to fold and function as independent domains In tetratricopeptide (TPR) repeats, the repeat unit is a helix-turn-helix motif The majority of TPR motifs occur as three to over 12 tandem repeats in different proteins The majority of TPR structures in the Protein Data Bank are of isolated domains Here we present the high-resolution structure of NlpI, the first structure of a complete TPR-containing protein We show that in this instance the TPR motifs not fold and function as an independent domain, but are fully integrated into the three-dimensional structure of a globular protein The NlpI structure is also the first TPR structure from a prokaryote It is of particular interest because it is a membrane-associated protein, and mutations in it alter septation and virulence Repeat proteins in general, and tetratricopeptide repeats (TPRs) in particular, have recently attracted interest from the perspectives of structure, function, folding and design [1–6] The TPR was first identified during sequence analysis of proteins CDC23 and nuc2+ from yeast [7,8], and has subsequently been found in a wide variety of polypeptides from all genera It is a degenerate 34-residue motif, which adopts a helix-turn-helix structure The first helix is usually termed the ‘A’ helix, while the second is referred to as the ‘B’ helix [6] The most common number of tandem TPRs within a single protein is three, but as many as 16 have been predicted on the basis of sequence analysis [2] Natural and designed TPRs whose structures have been determined share a common tertiary organization, which is dominated by interactions between A helices and the preceding AB pair (Fig 1A) Local AB, BA¢ and nonlocal AA¢ helix packing generate an extended superhelical array with right-handed twist The motif is often terminated by an additional A, or ‘capping helix’, whose exposed edge is hydrophilic in character, thus promoting favourable interactions with solvents TPRs are therefore distinct from globular proteins, because they not possess a single hydrophobic core Instead, stabilizing interactions are distributed throughout the structure One significant consequence of TPR superhelicity is the creation of concave (‘front-face’) and convex (‘backface’) surfaces In the best-characterized examples to date, the 3-TPR domains TPR1 and TPR2A of human Hsp organizing protein (HOP), which bind the C-terminal residues of Hsc70 and Hsp90, respectively, ligand recognition occurs at the concave front-face [9,10] Each of the 3-TPR domains of HOP behave as structurally and functionally independent domains in vitro In vivo, functioning independently as part of Abbreviations HOP, Hsp organizing protein; Hsp, heat shock protein; SUPR, superhelical peptide repeat; TPR, tetratricopeptide repeat 166 FEBS Journal 272 (2005) 166–179 ª 2004 FEBS C G M Wilson et al Fig Canonical TPR structure and NlpI sequences (A) Example of TPR extended helical structure, from the consensus design 1NA0.pdb [6] Three repeats are the most common number seen The AB and AA¢ W packing angles are responsible for curvature and superhelicity of the motif (B) Amino acid sequence of NlpI from the translation of the nlpI gene [22], including the signal prosequence (underscored) and lipobox cysteine modification site (boxed) Proposed TPR motifs are shaded grey [27] (C) Alignment of the putative NlpI TPRs compared to the signature motif Variations from the consensus are unshaded positions within the vertical shaded bars The fourth repeat contains the fewest matches to the consensus the full-length HOP, they act to facilitate the assembly of multichaperone regulatory complexes The structural independence of these TPR domains, and the presence of independent ligand-binding sites in each, has been assumed to be characteristic properties of TPR domains Methods that identify motifs from amino acid sequence (e.g pFAM [11]) readily predict TPRs, with the implication that they are discrete domains TPR domains, or even subsets of TPRs within a domain [12], are often studied independently In the course of a wider effort toward understanding TPR structure and function, a number of related observations intrigued us First, the only structures of FEBS Journal 272 (2005) 166–179 ª 2004 FEBS Crystal structure of NlpI natural TPRs in the PDB (13 TPR-containing coordinate sets) consist of nonglobular, extended arrays of helices [9,10,13–21] Second, the majority of these (11 structures) are for isolated domains, taken from larger parent proteins Third, of those structures that consist of more than the TPR sequence alone, the nonTPR component can be unstructured, a C-terminal capping helix [14], can bind back as an extended polypeptide to the concave face [16], or can assume a completely independent domain organization [19,20] Fourth, a single structure has been determined where TPR motifs are seen to fold back on themselves: in the seven-repeat peroxisomal targeting signal receptor PEX5, repeats and pack against repeats and through loop motifs [13] This is an unusual example, however, because an alternative conformation for PEX5 has been determined in which the structure is extended [18] This may represent a novel conformational switching mechanism where dynamic, longrange inter-TPR interactions are critical to function Alternatively, these two conformations could reflect different crystallization conditions Fifth, no structure of a prokaryotic TPR has yet been determined Here we describe the first structure of a complete TPRcontaining protein from Escherichia coli New lipoprotein I (NlpI) is a 32 kDa, 276 residue protein from E coli K-12 [22] The corresponding chromosomal gene encodes a 294 amino acid polypeptide, whose N-terminal 18 residues comprise a periplasmic export sequence and ‘lipobox’ motif (Fig 1B) Following translocation across the inner plasma membrane, the prosequence and lipobox cysteine are recognized, enzymatically modified and proteolytically processed by components of the lipoprotein biosynthetic pathway This yields an N-acyl-S-sn-1,2-diacylglyceryl-cysteine (residue 19) as the N-terminus of the mature, 276 residue, membrane-anchored protein [22] The identity of the residue at the +2 position (Ser20) in the mature protein suggests that NlpI is not retained at the inner membrane, but is likely to be anchored at the outer membrane [23–25] The precise topological location (periplasmic or extracellular face) is not known NlpI has been proposed to play a role in bacterial septation, or regulation of cell wall degradation during cell division [22] Disruption of the chromosomal copy of the nlpI gene, or plasmid-mediated overexpression of the protein, both lead to altered cell morphology and to osmotic sensitivity NlpI is of potential clinical interest, because loss of the nlpI gene affects the synthesis of pili and flagellae, leading to changes in extracellular adhesion properties which are correlated with an invasive, pathogenic phenotype [26] A BLAST search for similar sequences 167 Crystal structure of NlpI finds highly conserved homologs from well-known pathogenic species (see below) Initial studies of NlpI primary structure predicted three tandem TPR repeats [22] A fourth repeat, immediately following the third, and a fifth independent TPR, have also been suggested [27], although the similarity of the fourth repeat to the consensus is weak (Fig 1C) On the basis of these analyses, one might anticipate the presence of an independent, extended three-repeat This could account for approximately 40% of the mature sequence, while the structural character of the remaining polypeptide is unknown Several properties make NlpI an attractive target for structural characterization The gene can be obtained directly from laboratory strains of E coli, while its size and periplasmic localization suggest it is likely to be a single domain, capable of independent folding The single cysteine is responsible for tethering the protein to a plasma membrane; the absence of disulfide bridges or other structural cysteine, simplifies the protein chemistry by removing the need for reducing agents during purification and handling We therefore cloned and expressed NlpI, and determined the protein structure by X-ray crystallography The structure reveals a fold in which the TPR is not an independent domain, but is an integral part of a globular protein Results and Discussion Cloning and expression of NlpI The gene for NlpI was obtained by direct PCR amplification from E coli DH10B [28] The sequence corresponding to the mature polypeptide (residues 20–294, lacking the signal prosequence and Cys20) overexpresses exceptionally well in BL21 (DE3), with yields of  100 mgỈL)1 (Fig 2A) Purified mature NlpI is soluble to at least 200 mgỈmL)1 in 10 mm Tris ⁄ HCl pH 8.0, 10 mm NaCl To investigate the anticipated 3-TPR domain of NlpI, we subcloned residues 62–197 This region also expresses well, but in contrast to the mature polypeptide, the majority of protein is found in inclusion bodies (Fig 2A) Material purified from the lysate soluble fraction precipitated after elution off Ni-nitrilotriacetic acid agarose Alternative expression, purification, solubilization and refolding regimes were investigated, but we were unable to obtain soluble 3-TPR This result was surprising, as we had anticipated the 3-repeat to be an independent domain The insolubility of this region was the first indication that the 3-TPR might be participating in a more complex structure We 168 C G M Wilson et al therefore pursued characterization of only the mature polypeptide (20–294) Analytical gel filtration chromatography indicated that mature NlpI runs at nearly twice its anticipated size ( 56 kDa vs 31 kDa) This was not entirely unexpected, because an extended array of helices occupies a larger hydrodynamic radius than a globular molecule of similar polypeptide length However, calculation of the Matthews coefficient from initial crystallographic data indicated that a dimer was most compatible with unit-cell dimensions (solvent content  56%) This was further supported by in vivo formaldehyde crosslinking (data not shown) that trapped a species corresponding to dimeric NlpI Structure of mature NlpI We have solved the crystal structure of mature NlpI ˚ to 2.0 A Data collection and refinement statistics are shown in Table A region of the 2Fo-Fc electron density map for residues 158–162 is shown in Fig The protein crystallised in space group P212121 with two monomers in the asymmetric unit, related by a twofold axis of noncrystallographic symmetry running through the dimer interface We conclude that the contents of the asymmetric unit represent the biologically active protein The two chains together form an arrow-shaped structure, wider than it is deep (Fig 2B,C) N-termini of both molecules share a common point of origin, a feature compatible with membrane localization through N-terminal lipid anchors on both chains Table shows the secondary structure components, interhelix packing geometries, and the angle of rotation between the AB helix pairs present With the exception of an extended, but not unstructured region of polypeptide (30–37), NlpI is composed of a-helix (64%) and turn motifs (23%) NlpI monomers can be described generally as a superhelical array of helix-turn-helix motifs, in which the C-terminus is folded (rolled-up) inside the N-terminus (Fig 2D) A depression on one side of each monomer contains a bound Tris molecule This cavity, formed by the curvature and packing of helices, is highly suggestive of a ligand binding pocket and we speculate that it may represent the functional site of the protein Helix packing interactions TPRs Many features of the distribution of side chain contacts within NlpI are typical of a TPR protein The side chain contact map (Fig 4A) is dominated by a FEBS Journal 272 (2005) 166–179 ª 2004 FEBS C G M Wilson et al Crystal structure of NlpI A B C D Fig Solubility of NlpI constructs and the structure of mature NlpI (A) 10–20% gradient SDS ⁄ PAGE of NlpI expression products, showing the insolubility of the 3-TPR construct vs mature NlpI BenchMark molecular mass markers (lanes & 4); 3-TPR insoluble (lane 2, arrowhead) and soluble (lane 3); mature NlpI insoluble (lane 5) and soluble (lane 6, arrowhead); mature NlpI following TEV protease cleavage, and purification over Superdex 75 (lane 7, arrowhead, anticipated molecular mass of 31.8 kDa) (B) Side and (D) top views of the NlpI dimer Chains are coloured from N- (dark blue) to C-termini (orange) Axis of noncrystallographic rotational symmetry runs through the center ‘x’ (C) Monomer of NlpI, showing the rolled-up array of helices with the C-terminus folding within the curvature of the N-terminus Helix numbers are in brackets Note that ‘A’ helices locate to the globular center, and the perpendicular arrangement of helices 10 and 11, against helices and repetitive pattern of interactions parallel to the diagonal (i to i + 3, i to i + within a continuous helix), orthogonal to the diagonal (helix A interacting with helix B), then parallel to the diagonal (helix A interacting with helix A¢ of the next repeat), and finally returning to the diagonal (helix B interacting with A¢) This distribution is more or less continuous, reflecting a progression of helix-turn-helix AB, AA¢, BA¢ interactions through the structure The exception is helix 1, which interacts exclusively with helix through hydrophobic packing of bulky groups (e.g Leu44 against Leu77, Met47 against the aliphatic components of side chain Arg68) Of the six AB helix-turn-helix pairs (Table 2), four closely resemble FEBS Journal 272 (2005) 166–179 ª 2004 FEBS TPRs: helices and (TPR1), and (TPR2), and (TPR3) and 12 and 13 (TPR4) contain the characteristic pattern of signature residues that coincides with helix-loop-helix lengths Interhelix AB and AA¢ W packing angles fall within those typical for TPRs [6] These repeats correspond to the anticipated tandem 3-TPR, and the isolated fifth TPR predicted from the amino acid sequence (Fig 1C) [22,27] Helices and 12 (A helices of TPRs and 4, respectively) contain additional helical residues preceding the start of the TPR region The final helix (14) does not contain the solvating polar groups associated with terminating ‘capping’ helices found in the other TPR structures (e.g PP5, TPR1 and TPR2A of 169 Crystal structure of NlpI C G M Wilson et al ˚ Table SeMet-NlpI Data processing, MAD phasing and refinement statistics FOM, figure of merit, value from DM is at 2.0 A, whereas ˚ SOLVE ⁄ RESOLVE values are at 2.5 A Space-group P212121 ˚ Unit-cell (A) ˚ Wavelength (A) ˚ Resolution (A) a ¼ 64.35 b ¼ 81.65 c ¼ 136.66 Peak (0.9785) 30–2.05 408241 85218 96.1 (84.0) 33.8 (4.7) 5.4 (30.7) 4.8 0.58 0.72 0.64 17.5 ⁄ 20.6 4615 436 Total number of reflections Number of unique reflectionsa Completeness (%)a I ⁄ Sigmab Rmergeb (%) Redundancya FOM after SOLVE FOM after RESOLVE FOM after DM R ⁄ Rfree (%) Number of all atoms Number of water molecules ˚ Avearge B-factors (A2) Monomer A (main chain ⁄ side chains) Monomer B (main chain ⁄ side chains) Water molecules Ramachandaran plot (%) (most favoured ⁄ allowed ⁄ disallowed) ˚ rmsd bond lengths (A) rmsd angles (°) a Values are given with Friedel-pairs (hkl and -h-k-l) kept separate Remote (0.9500) 30–2.0 282547 91856 94.9 (81.2) 29.3 (2.7) 5.3 (46.6) 3.1 20.4 ⁄ 23.6 21.6 ⁄ 23.8 39.3 94.0 ⁄ 6.0 ⁄ 0.0 0.026 1.96 b Value for the high resolution shell is given in parentheses Fig Sample 2Fo-Fc density map for NlpI (sigma ¼ 1.0) Residues shown are Gln158-Asn162 (QDDPN) for chain A, which corresponds to the turn region between helices and Image was produced with BOBSCRIPT [57] and RASTER3D [58] HOP), because the majority of these residues participate in the protein core NonTPR helix motifs Packing interactions are more complex for the two remaining pairs of helices (8 and 9, 10 and 11) These are of particular significance since they are responsible for the compact structure of NlpI Helix pair 10–11 170 Inflection (0.9795) 30–2.0 506497 91502 96.0 (83.6) 40.4 (4.8) 4.9 (35.5) 5.5 (Fig 4B) is, at 27 residues (17 of which are helix) too short to be termed a TPR The interhelix AB W packing angle is the highest (+ 172°), bringing them close to parallel, and is also of the opposite sign to that which characterizes a TPR Interactions with the following pair of helices (12 and 13) is distinguished by the only negative AA¢ angle within the protein Critically, this combination of nonTPR packing angles imparts lefthanded superhelical character to the region The pitch of the overall right-handed superhelix is therefore reduced, which brings the C-terminus up toward the N-terminus Helices and correspond to the region of sequence postulated by some to be a fourth TPR, following on immediately after the tandem 3-repeat (Fig 1C [27]) However, the interactions taking place within this pair indicate that it is not a TPR Helix contains the hydrophobic sequence LWLYL(168–171), and these groups are involved in long-range interactions (discussed below) Helix participates more in these than in packing against its partner, helix The signature glycine residue of the A helix is missing, a space occupied in a TPR by a bulky hydrophobic or aromatic ring from the partner B helix (knobs-in-holes complementarity) Instead, the glycine position is occupied by alanine, with the remaining space filled by a tyrosine (Tyr171) from the same (A) helix The complementarity FEBS Journal 272 (2005) 166–179 ª 2004 FEBS C G M Wilson et al Crystal structure of NlpI Table Primary, secondary and tertiary structure statistics for mature NlpI Excluding the N-terminal 310 helix (H0), NlpI contains 14 a-helices W Packing angles characteristic of TPRs range from )160° to )174° (AB), 11° to 32° (AA¢) and 40° to 53° (AB-A’B¢ repeat rotation) [6] AB pairs and (helices 8–9, 10–11) not display true TPR characteristics, but are responsible for the sharp curvature of the array and reduced superhelical pitch, leading to the formation of a globular structure ˚ Helixa Residues Deviationb,c A AB pair W° c AB W° c Rotationd Helix pair sequence AA¢ (AB)(A¢B¢)° (signature TPR residues underscored) 27–29 38–51 58–74 78–91 – 6.4 7.6 5.9 – – (TPR1) – – )160.5 – – +31.3 – – 57.6 (pair 1–2) consensus motif W LG Y A F A P DDERAQLLYERGVLYDSLGLRALARNDFSQALAIRPDM 96–108 112–125 5.9 5.1 (TPR2) )165.5 +16.7 59.0 (pair 2–3) PEVFNYLGIYLTQAGNFDAAYEAFDSVLELDPTY 131–142 146–159 8.9 12.2 (TPR3) )153.0 +26.5 16.2 (pair 3–4) YAHLNRGIALYYGGRDKLAQDDLLAFYQDDPND 164–177 179–192 14.6 3.9 +163.3 +26.9 96.2 (pair 4–5) PFRSLWLYLAEQKLDEKQAKEVLKQHFEKSDKEQW 10 11 199–206 212–222 12.9 7.4 +172.6 )26.0 40.4 (pair 5–6) GWNIVEFYLGNISEQTLMERLKADATD 12 13 226–246 250–261 19.4 8.6 (TPR4) )158.3 +38.2 261 (pair 1–6) 14 269–283 7.9 – – – – a Defined by by PROCHECK [51] b From ideal helix geometry c NTSLAEHLSETNFYLGKYYLSLGDLDSATALFKLAVANNVHNF Calculated by PROMOTIF [52] d Obtained by transforming one helical pair onto another with lsqman [53] between helix and is therefore less marked, and they are less tightly packed together compared to AB pairs before or after (Fig 4B) The apparent lack of contacts between helices and is, however, compensated for by an unusual association with the next pair of helices, 10 and 11 These pack against and at an angle of 96° (visible in Fig 2D), which is the highest interrepeat rotation angle within the structure A unique, nonTPR interaction takes place where the indole ring of Trp200 (helix 10) inserts between helices and 9, against Phe190 and the amide backbone of Phe165 This locks the pairs together (Fig 4D) The abrupt increase in helical array curvature is the second factor responsible for bringing distal regions of sequence back toward proximal ones Long-range interactions & globularity The presence of long-range contacts within NlpI is revealed by clusters in the contact map far from the diagonal (Fig 4A) These interactions take place only between A helices, which dominate the inside of the NlpI helical roll (Fig 2C) The clusters can be considered as four overlapping groups (Fig 4E) Cluster involves helices 12 and 14, packing against the N-terminal region of NlpI These constitute the most distant interactions between elements of primary FEBS Journal 272 (2005) 166–179 ª 2004 FEBS structure, and include a hydrogen bond between the backbone carbonyl of Asn263 and the backbone amide of Leu34 The positive AA¢ W angle between helix 12 and helix 10 is in part responsible for this Cluster consists of loop against loop interactions between TPR1 and TPR2, with helix 14 (including a Ca backbone contact between Gly76 and His266) From functional perspectives this is perhaps significant, as this first TPR is more open than any other, forming an exposed ‘lip’ on the NlpI monomer, and therefore most closely resembles classical TPR front-face environments in providing an interaction surface Cluster contains solvent inaccessible hydrophobic groups (Leu134, Ile138, Val269) and hydrogen bond interactions (Tyr142-Lys242) between the third and fourth TPR, with helix 14 Cluster 4, which overlaps with clusters and 3, forms the core of NlpI long-range interactions These include bulky hydrophobic, aromatic and surface solvent exposed groups from helices 6, 8, 12 and 14 For example, Trp169 (helix 8) has hydrophobic contacts with Leu134, Ile138, Tyr142 (helix 6), Phe238 (helix 12) and Val269 (helix 14) Trp169 and Tyr273 (helix 14) are on opposite sides of the protein core, and not interact directly, but they are bridged by aliphatic and aromatic groups (Ile138, Tyr142, Phe238 and Val269) from helices and 12 Aromatic ring interactions, including ‘T’ face-edge (Tyr131-Phe156, 171 Crystal structure of NlpI A C G M Wilson et al B D E C F Fig Contact map of mature NlpI and packing interactions (A) Backbone (upper left from diagonal) and side chain (lower right from diago˚ nal) contacts within A Long-range contact clusters are boxed (B) Packing interactions between nonTPR helices 10 (red) and 11 (blue), and (C) helices (red) and (blue) Space-filling atoms shown are large and small hydrophobic residues (F, Y, W, I, L, V, A and G) Bulky groups of helix point toward the protein core (D) View of NlpI helices and (with helix removed), showing diminished association between the pair, and the insertion of Trp200 from helix 10 Right-handed superhelical curvature imparted by the first three TPRs appears to cease, allowing the subsequent structure to roll-up (C) Location of long-range packing clusters from (A), which define the core of NlpI (F) Aromatic and bulky side chains surrounding Trp169 (orange) Tyr141-Tyr142, Phe205-Tyr243) and offset pi-stacking (Phe85-Tyr101, Trp298-His232) are evident, with the spaces between these moieties filled by bulky aliphatics (Fig 4F) Long-range interactions are a characteristic property of globular protein structures By virtue of their extended helical organization, TPR and other repeat proteins typically lack this feature, but rather have stabilizing apolar contacts distributed throughout the molecule, both within and between repeats In contrast, NlpI contains a central hydrophobic core, composed of distant motifs from TPR and nonTPR helices TPRs are therefore compatible with globular structures, but they not appear to be capable of defining it The increased curvature and reduced superhelical pitch required to form a compact, tertiary structure are derived from nonTPR elements within the fold Quaternary organization NlpI is a homodimer in solution, and the crystal structure reveals monomers to be related by twofold axis of symmetry The dimer interface consists of the extended N-terminal region, helix and TPR helices 2, 3, 11, 12, 13 and 14 (Table 3, Figs and 6B) The values obtained for interface surface area, interaction 172 type (two-thirds hydrophobic, but also hydrogen bonds and salt-bridges), gap volume index and planarity (which relate to the complementarity of the interface surfaces) fall within the ranges associated with known homodimeric states [29] Three aspects are especially noteworthy First, rotational symmetry places the N-termini of both monomers spatially close to each other A lipid-modified dimer will therefore be anchored to a plasma membrane in a specific orientation (N-termini ‘face down’ toward the membrane) This is significant, because the potential ligand binding Table NlpI dimer interface statistics Values were obtained with SURFNET [29,55] SA ¼ surface area Interface statistics Region Residues ˚ Monomer buried SA (A2) 1585.5 % Monomer SA 12.6 % Nonpolar atoms % Polar atoms Planarity Hydrogen bonds Salt bridges Buried H2O Gap volume index N-terminal 23–26, 30–36 Helix 38, 41, 44, 45, 48, 49, 51 36.6 Helix 68, 76, 77 63.4 Helix 78, 79, 80, 83 2.5 Helix 11 212, 216 16 Helix 12 237, 240, 244 Helix 13 255, 258, 259,261, 262, 264, 266 16 Helix 14 271, 275, 278 2.45 Total 38 FEBS Journal 272 (2005) 166–179 ª 2004 FEBS C G M Wilson et al Crystal structure of NlpI pendent domain One could therefore ask if there are any features of the TPR sequences that hint at differences between these TPRs, and those that fold independently Unfortunately, with the limited sequence– structure data available at this stage, there are no correlations strong enough to allow us to predict, or subclassify, which TPR sequences will form an extended array and which will adopt globular structures Implications for function: a putative binding cleft Fig NlpI dimer interface Chain B has been translated and rotated to expose the surface in contact (yellow) The interface is composed of remote regions of sequence from the N- and C-termini Val32 is indicated to illustrate the rotational symmetry of the interface Contacts were obtained with SURFNET [55] and CONTACT from the CCP4 [42,43] suite of programs cleft (discussed below) of each monomer would then be exposed, and oriented roughly perpendicular to the plane of the membrane NlpI localized in this manner could then serve as a tether to which other functional components would bind Second, the dimer interface is made up of distant regions of monomer sequence That is, N-terminal and C-terminal portions of monomer polypeptide come together, forming the molecular surface Quaternary structure is therefore dependent on tertiary structure, and their formation may be interdependent (cooperative) Third, it was previously noted that the first TPR (helices and 3) participates in long-range interactions within a monomer through loop residues (Asp73, Ser74, Leu75, Arg78), while the majority of the inner front-face assumes an open ‘lip’ conformation (Fig 4D) In contrast, seven residues of the outer back-face participate in the dimer interface, packing against C-terminal portions of polypeptide from the partner molecule Consideration of monomeric NlpI alone gives the impression that these helices make few molecular contacts when in fact they make many, albeit with a separate polypeptide chain The insolubility of NlpI 3-TPR (fragment 62–197) is therefore understandable, in terms of the failure of an isolated motif to form critical intra- and intermolecular contacts These observations demonstrate the capacity, and on occasion the necessity, of TPRs to participate at all levels of structure organization, and suggest that the fold is more versatile than was previously thought We now know the structure of NlpI, and observe that the TPRs in this protein not form an indeFEBS Journal 272 (2005) 166–179 ª 2004 FEBS We examined the conservation of NlpI structure through a sequence alignment of the 12 most similar sequences identified through a BLAST search (Fig 6A) When conserved positions are mapped onto the structure of NlpI, they correlate to three distinct locations within the protein Two of these are clearly structural in nature: the globular core (discussed previously) and the dimerization interface (also predominantly hydrophobic, Fig 6B), suggest that NlpI homologs share a common tertiary and quaternary organization The third conserved region corresponds to the depression on one side of each NlpI monomer (Fig 6C) The cleft is lined with polar (Asn267), acidic (Asp163, Glu235, Glu231 and Glu270), aromatic (Tyr131, Phe165, Trp198, Phe268) and hydrophobic groups (Ile104, Val269) Visually, the shape of the depression is highly suggestive of a binding site The presence of four invariant acidic groups (one aspartate and three glutamate) implicates electrostatic interactions, possibly with a basic motif, in the putative binding event The high degree of sequence conservation in the cleft suggests all homologs of NlpI bind the same ligand In addition, our attention was drawn to this cavity during the final stages of model building, because it contained a patch of 2Fo-Fc density that could not be accounted for by the polypeptide, water molecules, Mg2+ or Cl– ions The structure of Tris, also a component of the crystallization mother liquor, was found to fit the density envelope, making hydrogen bonds with carboxylate groups of Glu235 and Glu270, and with the back bone amide of Val269 Phe165 and Phe268 face each other, flanking the two carboxylates and Tris (Fig 6D) NlpI is thought to play a role in the regulation of the cell wall and extracellular surface, but its exact function is not known, and no ligand interactions have yet been described There has been some suggestion that the C-terminus may associate with the periplasmic protease Tsp, and it has been proposed that removal of residues beyond Gly282 serves to activate the protein [27] However, the C-terminus of NlpI does not contain a motif that resembles the canonical ‘WVAAA’ associated with 173 Crystal structure of NlpI C G M Wilson et al A B C D Fig Homologs of NlpI, structural conservation the putative binding cleft (A) CLUSTALX alignment [48] of the 12 sequences most similar to E coli NlpI identified by BLAST [49] Positions are colored as follows: red, identical (*); yellow, similar (:) 1, Escherichia coli; 2, Salmonella typhinurium; 3, Yersinia pestis; 4, Yersinia enterocolitica; 5, Vibro haemolyticus; 6, Vibro vulnificus; 7, Vibro cholerae; 8, Photorhabdus luminescens; 9, Photobacterium profundum; 10, Haemophilus influenzae; 11, Haemophilus ducreyi; 12, Shewanella oneidensis E coli residues underscored locate to the hydrophobic core (B) Molecular surface revealing conserved positions within the dimerization interface (mostly hydrophobic) (C) The surface of an NlpI monomer, showing the putative ligand binding cleft and bound Tris molecule (D) Tris molecule, conserved acidic and aromatic side chains within the cleft Orange dashes indicate hydrogen bonds between Tris and the amide backbone of Val269, and conserved side chain carboxylates of Glu235 and Glu270 Tsp recognition [30] Our structure suggests the functionality of NlpI in fact lies within the cleft associated with globular body of the fold Structural homologs A DALI search for homologous structures finds two PDB entries with significant similarity to NlpI The first, p67phox from human (Z-score ¼ 12.4, rmsd of ˚ 2.5 A over 152 residues) consists of four TPR motifs, and an extended C-terminus that packs against the concave front-face groove through hydrophobic 174 interactions The intracellular ligand, Rac-GTP, is required for the assembly of the multiprotein NADPH oxidase complex, and binds to the surface formed by TPR connecting loops and the C-terminal polypeptide [16] The ligand-binding mode is therefore distinct from that of TPR1 and TPR2A of HOP The structural similarity between NlpI and p67phox is illustrated in Fig 7A, and reveals a close match between the first four AB repeats of each protein The second structure, domain III from E coli maltose transcriptional regulator MalT (Z-score ¼ 12.3, FEBS Journal 272 (2005) 166–179 ª 2004 FEBS C G M Wilson et al Crystal structure of NlpI an alternate arrangement, in which the TPR is inseparably part of the whole structure Nothing about the sequence, or any a priori considerations, suggested that this structure would be different from the TPRs from independent domains The structure provides a strong hint at the location of the active site, though as yet the ligand bound by NlpI has not been identified Its involvement in bacterial virulence, and likely presence of identical interactions in many pathogenic species, makes NlpI a potential target for new antibiotics Experimental procedures Fig Structural homologues of NlpI Structure alignment of NlpI (blue) with (A) p67phox and (B) domain III of MalT (yellow) Superimposition of coordinates was performed with LSQ_EXPLICIT and LSQ_IMPROVE [45] ˚ rmsd of 4.3 A over 212 residues), consists of eight superhelical peptide repeat (SUPR) motifs that assume a superhelical fold [31] SUPRs resemble TPRs, but their helices are slightly longer (16–18 residues) and the sequence consensus is more degenerate The N-terminal region of MalT is responsible for functional dimerization, while the C-terminus is thought to contain a maltotriose binding site, formed by the concave surface of four SUPR repeats Close structural similarity can be seen between the first three AB repeats of NlpI and MalT (Fig 7B) However, by the sixth repeat the reduction in NlpI superhelical pitch has folded the protein back onto itself, while MalT continues in a more regular superhelix (and in consequence lacks hydrophobic core interactions) In terms of their biological roles, p67phox and MalT both mediate intermolecular interactions, and are responsible for the assembly of multiprotein complexes It is therefore interesting to speculate, on the basis of structural identity and the presence of a conserved surface cleft, whether NlpI participates in analogous multiprotein assemblies in E coli Conclusion We have determined the first structure of a complete TPR-containing globular protein, which is also the first TPR from a prokaryotic organism The structure reveals an intimate association between the TPR motifs and the rest of the protein, showing how the TPR participates in the overall fold Until now, many of the TPR-containing regions of proteins have behaved as separate domains: they fold and function completely independently of the rest of the protein Here we show FEBS Journal 272 (2005) 166–179 ª 2004 FEBS Cloning NlpI constructs DNA encoding NlpI sequences was obtained by PCR amplification from a single colony of E coli DH10B [28] (Invitrogen, Carlsbad, CA, USA), grown on Luria–Bertani agar overnight All oligonucleotides were chemically synthesized by the W M Keck Core Facility (Yale University, New Haven, CT, USA) Primers to amplify mature NlpI (residues 20–294) were 5¢-aataatccatggggagtaatacttcctggcgta aaagtgaagtcc-3¢ and 5¢-attattggatccctattgctggtccgattctgccag-3¢ 3-TPR NlpI primers (residues 62–197) were 5¢-aataatccatgg gggcacagcttttatatgagcgcggag-3¢ and 5¢-aataatggatcctcactgttc cttatccgatttttcgaagtgc-3¢ PCR products were doubly digested with NcoI and BamHI (New England Biolabs, Beverly, MA, USA), and purified by agarose gel electrophoresis onto dialysis membrane, prior to ligation into doubly digested, dephosphorylated expression vector pET11a-HT This vector was assembled in-house from vectors pProEX-HTa (Invitrogen) and pET11a [32] (Novagen, San Diego, CA, USA), and places cloned sequences under T7 promoter control Expression in an E coli DE3 bacterial host produces an N-terminal hexahistidine-tagged protein, cleavable with TEV protease Ligation products were transformed into electrocompetent E coli DH10B (Invitrogen), and transformants sequenced by the W M Keck Facility Expression and purification Plasmids, verified by DNA sequencing, were transformed into E coli BL21 (DE3) Gold (Stratagene), and grown in Luria–Bertani medium supplemented with 100 lgỈmL)1 carbenicillin at 37 °C until cell culture absorbance at 600 nm was 0.5 The temperature was reduced to 25 °C before induction with 100 lm isopropyl thio-b-d-galactoside Cells were harvested by centrifugation (6000 g, 20 min) after h further growth, and stored at )80 °C Selenomethione (SeMet)-labelled NlpI was expressed in E coli methionine auxotroph B834 (DE3), grown in M9 medium [33] supplemented with 50 mgỈL)1 l-methionine and 100 mgỈL)1 thiamine At a cell density of D600 ¼ 0.4, bacteria were harvested and resuspended in fresh M9 supplemented with 175 Crystal structure of NlpI 50 mgỈL)1 l-selenomethione (Pierce, Rockford, IL, USA) Growth was continued for 20 at 25 °C, before induction with 100 lm isopropyl thio-b-d-galactoside overnight Cell pellets were resuspended in buffer A [50 mm Tris ⁄ HCl pH 8.0, 300 mm NaCl, 0.1% (v ⁄ v) Triton X-100] plus lysozyme to a final concentration of 0.1 mgỈmL)1, and incubated for 30 on ice prior to sonication (20 second pulses at maximum power, for a total process time of min, with 20 s cooling between pulses) Insoluble material was removed by centrifugation (20 min, 26 000 g, °C) Affinity purification was performed with slow rocking at °C overnight, with one-fifth volume of Ni-charged nitrilotriacetic acid agarose slurry (Qiagen) Washing steps were performed at room temperature in a disposable standing column (Bio-Rad, Hercules, CA, USA), with bed volumes buffer A and bed volumes buffer B (50 mm Tris ⁄ HCl pH 8.0, 300 mm NaCl, mm imidazole) Bound protein was eluted in buffer C (50 mm Tris ⁄ HCl pH 8.0, 150 mm NaCl, 300 mm imidazole) Hexahistidine tags were removed by treatment with 10 units of AcTEV protease (Invitrogen) overnight at room temperature, followed by dialysis against buffer D (50 mm Tris ⁄ HCl pH 8.0, 150 mm NaCl) TEV protease and uncleaved fusion were removed by gravity flow through a mL bed of fresh Ni-nitrilotriacetic acid agarose Protein was loaded onto a Superdex S200 16 ⁄ 60 prep grade column (Amersham Biosciences), equilibrated in buffer D Fractions containing NlpI were pooled, dialysed against buffer E (10 mm Tris ⁄ HCl pH 8.0, 10 mm NaCl) and concentrated with a Centriprep YM10 spin concentrator (Millipore, Billerica, MA, USA) Mature NlpI mass was verified by MALDI mass spectrometry Protein concentration was estimated by SDS ⁄ PAGE against BenchMark Protein Ladder (Invitrogen), and by absorbance at 280 nm assuming a calculated emature NlpI ¼ 43 240 m)1Ỉcm)1 [34] Size exclusion chromatography Analytical sizing runs were performed with a Superdex 200 10 ⁄ 300 GL Tricorn column (Amersham Biosciences) equilibrated in buffer D Flow-rate was 0.25 mLỈmin)1 and detection was by UV absorbance at 280 nm Calibration runs were performed with Gel Filtration Standard mix (Bio-Rad) Purified NlpI was diluted in buffer D to give 100 lL at concentrations between 15 lm and 1.5 mm (0.5 and 50 mgỈmL)1) The apparent molecular mass was estimated from proteins standards, assuming an inverse relationship between retention time and log molecular mass Crystallization of mature NlpI Purified NlpI was subjected to Crystal Screen I and II (Hampton Research, Aliso Viejo, CA, USA) as hanging drops, at a protein concentration of 150 mgỈmL)1 Crystals 176 C G M Wilson et al formed overnight in 100 mm Hepes pH 7.5, 200 mm MgCl2, 30% (v ⁄ v) PEG 400 at 22 °C Final conditions were 50 mgỈmL)1 NlpI mixed : with 100 mm Tris ⁄ HCl pH 8.5, 360 mm MgCl2, 27% (v ⁄ v) PEG 400, 0.8% (v ⁄ v) n-butanol, at 22 °C SeMet-labelled NlpI crystallized isomorphously, under identical conditions Rectangular crystals grew over days at 22 °C to 0.3 · 0.5 · 1.0 mm Data collection & phasing Crystals were flash-frozen from mother liquor in a nitrogen gas cryo-stream In-house data collection used a Mar345 image plate detector (MAR Research), coupled to a Rigaku CuKa rotating anode source NlpI crystallized in the space˚ group P212121, with unit-cell dimensions a ¼ 64.35 A, b ¼ ˚ ˚ 81.65 A, c ¼ 136.66 A, with two monomers in the asym˚ metric unit and a Matthews coefficient of 2.82 A3ặDa)1 (solvent content ẳ 56.36%) [35,36] A three-wavelength MAD data set was collected from a SeMet–NlpI crystal at beamline X12C, of the National Synchrotron Light Source, Brookhaven National Laboratory Data was collected for three wavelengths (Table 1), in two oscillation ranges from / and / +180° to ensure both Friedel-mates were collected for each reflection and to obtain accurate, redundant anomalous data However, less data was collected for the remote wavelength because of limited beam time Anomalous data was nevertheless complete for the remote wavelength ˚ ˚ (Table 1) Data from 30 A to 2.0 A were indexed and scaled with the hkl2000 software package [37] (Table 1) The search for Se sites was performed with solve [38] at ˚ 2.5 A, assuming a dimer containing eight Se atoms within the asymmetric unit-cell (including an N-terminal SeMet) Six Se sites were found (N-terminal SeMet was disordered) The initial phases from solve were subjected to solvent flattening and twofold noncrystallographic symmetry (NCS) averaging The initial model, containing most of the secondary structure elements, was obtained with resolve [39] at ˚ ˚ 2.5 A However, phase extension to 2.0 A did not work as well as expected Solvent flattening, NCS averaging with an operator (obtained from the resolve dimer model, and refined with imp [40]), and phase extension, were repeated with dm [41] in 100 steps, using the CCP4 graphical user interface [42,43] and the phases from solve This resulted in significant improvement in map quality Model refinement Several schemes were tested for refinement The best results were obtained using cns [44] for rigid body refinement at ˚ ˚ 2.0 A, and conjugate gradient minimization at 2.5 A with maximum likelihood target, resulting in R-factors of R ⁄ Rfree ¼ 30.0 ⁄ 30.9% The model was then manually improved and completed in o [45] Thereafter the structure was refined with refmac 5.0 [46] through the ccp4 graphical user interface [42,43], with overall anisotropic B-factor FEBS Journal 272 (2005) 166–179 ª 2004 FEBS C G M Wilson et al ˚ refinement of data and bulk solvent to 2.0 A resolution The first round of refinement converged to R-factors of R ⁄ Rfree ¼ 21.1 ⁄ 25.2% The model was once more inspected and refined further with refmac 5.0 In final rounds of refinement water molecules were added with arp ⁄ warp [47], and manual inspection with o Initially, strict NCS restraints were used, but these were released in the final stages of refinement (‘loose’ in refmac ⁄ CCP4) The final model contained residues 26–284 for chain A, residues 23–284 for chain B, 436 water molecules in asymmetric unit and one bound Tris molecule per monomer, with R-factors R ⁄ Rfree ¼ 17.5 ⁄ 20.6% and excellent geometry (Table 1) Sequence and structure analysis Sequence alignments were performed with clustalx [48] NlpI sequences were obtained from, and BLAST searches [49] performed through, the ExPASy database server (http://www.expasy.org/) [50] Stereochemical quality of the model was assessed with procheck [51] Helix geometry and packing angles were calculated with promotif [52], and the CCP4 utility lsqman [53] Main chain and side contacts were determined using contact within CCP4 [42,43] Contact map figures were generated with molmol [54] The dimer interface was assessed surfnet [55] through the Protein–Protein Interaction Server (http:// www.biochem.ucl.ac.uk/bsm/PP/server/) at UCL, London, UK [29] Structural homologs were identified through the DALI server (http://www.ebi.ac.uk/dali/) at the European Bioinformatics Institute, Hinxton, UK [56] Electron density map images were created with bobscript [57], and rendered with raster3d [58] Structure representations were prepared with pymol (DeLano Scientific, http://pymol sourceforge.net) Model coordinates Coordinates and structure factors have been submitted to the RCSB Protein Databank, under accession code 1XNF.pdb Acknowledgements This work was supported in part by NIH grants GM62413 and GM57265 (L.R.) 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gggcacagcttttatatgagcgcggag-3¢ and 5¢-aataatggatcctcactgttc cttatccgatttttcgaagtgc-3¢... (Finland) Data for this study were measured at beamline X12C of the National Synchrotron Light Source We are grateful for the assistance of Dr Anand Saxena in the use of this beamline The National... chemically synthesized by the W M Keck Core Facility (Yale University, New Haven, CT, USA) Primers to amplify mature NlpI (residues 20–294) were 5¢-aataatccatggggagtaatacttcctggcgta aaagtgaagtcc-3¢