Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus
1998; Springer Nature; Volume: 17; Issue: 21 Linguagem: Inglês
10.1093/emboj/17.21.6155
ISSN1460-2075
AutoresGabriel E. Soto, Karen Dodson, D. Ogg, Christopher Liu, John Heuser, Stefan D. Knight, Jan Kihlberg, C. Hal Jones, Scott J. Hultgren,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle2 November 1998free access Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus Gabriel E. Soto Gabriel E. Soto Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Karen W. Dodson Karen W. Dodson Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Derek Ogg Derek Ogg Structural Chemistry N62.6, Pharmacia and Upjohn, 112 87 Stockholm, Sweden Search for more papers by this author Christopher Liu Christopher Liu Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author John Heuser John Heuser Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Stefan Knight Stefan Knight Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Department of Molecular Biology, P.O. Box 590, S-751 24 Uppsala, Sweden Search for more papers by this author Jan Kihlberg Jan Kihlberg Department of Organic Chemistry, Umeå University, S-901 87 Umeå, Sweden Search for more papers by this author C.Hal Jones C.Hal Jones SIGA Pharmaceuticals, Inc., Research and Development Division, 4575 SW Research Way, Suite 230, Corvallis, OR, 97333 USA Search for more papers by this author Scott J. Hultgren Scott J. Hultgren Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Gabriel E. Soto Gabriel E. Soto Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Karen W. Dodson Karen W. Dodson Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Derek Ogg Derek Ogg Structural Chemistry N62.6, Pharmacia and Upjohn, 112 87 Stockholm, Sweden Search for more papers by this author Christopher Liu Christopher Liu Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author John Heuser John Heuser Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Stefan Knight Stefan Knight Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Department of Molecular Biology, P.O. Box 590, S-751 24 Uppsala, Sweden Search for more papers by this author Jan Kihlberg Jan Kihlberg Department of Organic Chemistry, Umeå University, S-901 87 Umeå, Sweden Search for more papers by this author C.Hal Jones C.Hal Jones SIGA Pharmaceuticals, Inc., Research and Development Division, 4575 SW Research Way, Suite 230, Corvallis, OR, 97333 USA Search for more papers by this author Scott J. Hultgren Scott J. Hultgren Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Author Information Gabriel E. Soto1, Karen W. Dodson1, Derek Ogg2, Christopher Liu1, John Heuser3, Stefan Knight4, Jan Kihlberg5, C.Hal Jones6 and Scott J. Hultgren1 1Department of Molecular Microbiology Washington University School of Medicine, St Louis, MO, 63110 USA 2Structural Chemistry N62.6, Pharmacia and Upjohn, 112 87 Stockholm, Sweden 3Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, 63110 USA 4Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Department of Molecular Biology, P.O. Box 590, S-751 24 Uppsala, Sweden 5Department of Organic Chemistry, Umeå University, S-901 87 Umeå, Sweden 6SIGA Pharmaceuticals, Inc., Research and Development Division, 4575 SW Research Way, Suite 230, Corvallis, OR, 97333 USA The EMBO Journal (1998)17:6155-6167https://doi.org/10.1093/emboj/17.21.6155 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone–subunit and subunit–subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site. Introduction The assembly of bacterial pili represents one of the best-characterized model systems available for the study of macromolecular assembly. These adhesive organelles play a crucial role in the pathogenesis of many virulent bacterial strains, as they mediate binding between the invading organisms and complementary receptors on the surfaces of host cells (Hultgren et al., 1996). Their assembly requires the ordered progression of multiple protein–protein recognition events, which in many cases are orchestrated by the ubiquitous chaperone–usher pathway. This pathway is involved in the assembly of >25 of these and other non-pilus organelles of attachment, and its study has provided insight into the basic biological processes of chaperone-assisted import, folding and targeting of proteins to specific assembly sites (Hung et al., 1996). The key features of this pathway are well illustrated by the prototypical P pili. These organelles are expressed in up to 90% of uropathogenic strains of Escherichia coli isolated from patients with pyelonephritis, versus only 5–10% of human fecal E.coli isolates (Hultgren et al., 1996). Encoded by genes of the pap operon, P pili are oligomeric structures containing a distal tip fibrillum attached to a pilus rod (Kuehn et al., 1992). The tip fibrillum is comprised of repeating PapE subunits and contains the PapG adhesin at its distal end, which is thought to be connected to the tip via the PapF adaptor protein (Jacob-Dubuisson et al., 1993). The pilus rod is made up exclusively of PapA subunits arranged in a right-handed helical cylinder and is thought to be connected to the tip fibrillum via the PapK adaptor (Jacob-Dubuisson et al., 1993). PapH appears to terminate pilus assembly and is important for the proper anchoring of the rod to the bacterial cell's outer membrane (Baga et al., 1987). Pilus assembly in vivo also requires the expression of two additional proteins encoded by the pap operon: the periplasmic chaperone PapD and the outer membrane usher PapC. The latter forms an oligomeric pore (Thanassi et al., 1998) in the outer membrane that regulates pilus assembly at least in part through kinetic partitioning of the chaperone–subunit pre-assembly complexes, with preferential binding of the chaperone–adhesin complex serving to initiate pilus assembly (Dodson et al., 1993; Saulino et al., 1998). PapD is the prototypical member of a highly conserved family of chaperones that are required for the assembly of both pilus and non-pilus adhesive organelles (Holmgren et al., 1992; Hung et al., 1996). PapD is known to play a multifunctional role in pilus assembly: (i) it mediates the partitioning of nascently translocated subunits out of the inner cytoplasmic membrane and into the periplasm; (ii) it is involved in the folding of nascent subunits into a native-like conformation; and (iii) it prevents the premature aggregation of subunits within the periplasmic space, which is otherwise toxic to the bacterial cell (Jones et al., 1997). However, the lack of any data at atomic level resolution on the structure of these hetero-oligomeric organelles or of a complete chaperone–subunit complex, together with the inability to obtain stable subunits in the absence of chaperone, has impeded attempts to determine precisely how chaperones carry out these functions in vivo. At present, it is known that periplasmic chaperones like PapD form complexes with pilus subunits prior to their assembly. Several lines of evidence strongly indicate that chaperones recognize and bind to a highly conserved C-terminal motif present in all pilus subunits assembled by PapD-like chaperones (Figure 1A). This motif is characterized by a series of alternating hydrophobic residues flanked by a glycine located 14 residues upstream from the C-terminus and a penultimate tyrosine. The evidence that chaperones bind to this region includes: (i) the finding that deletion of this C-terminal region abolishes chaperone–subunit complex formation (Hultgren et al., 1989); (ii) the demonstration that PapD specifically binds synthetic peptides whose sequences correspond to the C-terminal region of P pilus subunits; and (iii) the co-crystallization of PapD bound to a peptide corresponding to the last 19 amino acids of the PapG adhesin (Kuehn et al., 1993). Figure 1.(A) Conserved C-terminal motif present in pilus subunits assembled by the PapD chaperone. For a comprehensive alignment of this region among all adhesive organelle subunits known to be assembled by members of the immunoglobulin-like chaperone superfamily, see Hung et al. (1996). Key: yellow boxed residues, conserved alternating hydrophobic residues; blue boxed residues, conserved glycine at position 14 from the C-terminus. (B) Conserved N-terminal motif present in pilus subunits assembled by the PapD chaperone. Key: yellow boxed residues, conserved alternating hydrophobic residues; blue boxed residues, conserved glycine; pink boxed residues, conserved cysteine. Download figure Download PowerPoint In this study, we elucidated the molecular basis by which periplasmic chaperones orchestrate the assembly of these hetero-oligomeric organelles. In a series of complementary experiments, we characterized the structural aspects of chaperone–subunit recognition in the pre-assembly complexes and subunit–subunit interactions in the assembled pilus structure. The data argue for a model in which the conserved chaperone-binding motifs of subunits are mapped to one or more assembly surfaces that participate in quaternary subunit–subunit interactions after chaperone uncapping. Furthermore, the formation of these assembly surfaces may be coupled to their capping and subsequent protection from premature assembly following chaperone-mediated extraction of the subunits from the inner membrane. Results Effect of C-terminal mutations on the stability of PapG–PapF and PapG–PapD complexes In order to investigate the possibility that the conserved C-terminal motif of pilus subunits is involved in mediating subunit–subunit contacts, we used site-directed mutagenesis to modify the pattern of alternating hydrophobic residues at positions 307, 309, 311 and 313 of the PapG adhesin (corresponding to positions 8′, 6′, 4′ and 2′, respectively, counting from the C-terminus of the protein). We examined the effects of 15 distinct point mutations within this region with respect to PapG–PapF interactions in the mature pilus, as measured by the relative temperature required for dissociation of PapG from PapF in purified pilus tips (Figure 2). The results of these experiments are summarized in Table I. Generally speaking, mutations at positions M307, M309 and L311 of PapG reduced PapG–PapF complex stability (i.e. a lower temperature was required for the complete dissociation of the variant PapGs from PapF relative to the wild-type adhesin), whereas mutations at position F313 had little or no effect on PapG–PapF interactions. Since the side chains at every other position in a β-strand are oriented in the same direction, these results suggest that the side chains of PapG residues 307, 309 and 311 constitute part of a continuous assembly surface that interacts with a complementary assembly surface on PapF. The absence of any discernible effect at position 313 despite the array of substitutions explored suggests that this site is not critical for PapG–PapF interactions, although it may still contribute to the subunit–subunit interface. The requirement that cells be able to produce intact tip fibrillae for purification precluded the use of this assay to study the effects of mutations in the C-terminal region of the other tip subunits, PapE and PapF; the former is the main component of the tip polymer, while the latter has been shown to be required for the nucleation of tip assembly (Jacob-Dubuisson et al., 1993). Figure 2.(A) Sequence of the C-terminal region of the PapG adhesin and summary of site-directed mutants used to examine PapG–PapF interactions. Underlined positions are conserved across other pilus subunits. (B) Tip fibrillae containing either wild-type PapG or one of 15 point mutants were prepared and incubated in SDS loading buffer for 10 min at incremental temperatures prior to analysis by SDS–PAGE. Shown here are the data for tips isolated from cells expressing wild-type PapG. As seen in this Coomassie Blue-stained gel, bands corresponding to PapG–PapF complexes and higher order PapK–(PapE)n–PapF–PapG assemblies were observed at incubation temperatures up to 65°C. At 71°C and above; however, the PapG–PapF and higher order complexes were completely dissociated, releasing PapG as a clearly resolved monomer. The identity of the PapG band was confirmed by immunoblotting with α-PapG-specific antisera. The identity of the PapF–PapG band was confirmed by its excision from a duplicate gel and subsequent analysis by SDS–PAGE and immunoblotting with α-tip antisera following incubation at 95°C. Download figure Download PowerPoint Table 1. Effects of PapG C-terminal mutations PapG variant PapF–G complex stabilitya PapD bindingb Wild-type ▪▪▪▪ ●●●● M307S ▪▪▪ ● M307T ▪▪▪ ● M307P N/A ●● M309A ▪▪▪▪ ● M309S ▪ ● M309P N/A ● L311A ▪▪▪ ● L311S ▪▪ ● L311T ▪▪▪ ●● F313G ▪▪▪▪ ● F313L ▪▪▪▪ ●●● F313M ▪▪▪▪ ●●● F313A ▪▪▪▪ ● F313V ▪▪▪▪ ●● F313N ▪▪▪▪ ● a Relative stability of PapG–PapF interaction compared with wild-type PapG, as measured by the temperature required to achieve complete dissociation of PapG–PapF complexes. Each block (▪) represents the number of temperature increments (shown in Figure 2B) above 42°C required for complete dissociation [e.g. four blocks indicate that all PapG ran as a monomer by lane 8 (71°C)]. ‘N/A’ signifies that enough material could not be purified from the specified construct in order to perform the experiment. b Relative binding to PapD compared with wild-type PapG, as measured by ELISA using a PapG–MBP fusion protein. Each dot (●) represents ∼25% binding capacity of wild-type at the highest concentration studied. We also examined the effects of these mutations on PapG–PapD interactions using an in vitro enzyme-linked immunosorbent assay (ELISA). Maltose-binding protein–PapG fusions (MBP–PapG140) were constructed where the C-terminal 140 amino acids of PapG, containing each of the C-terminal variants, were fused to the C-terminus of MBP. The wild-type MBP–PapG140 fusion has been shown previously (via this assay) to bind PapD with affinity roughly equal to that of full-length PapG (Xu et al., 1995). In general, mutations at positions M307, M309, L311 and F313 of the PapG construct significantly reduced PapD binding (Table I). This finding is consistent with the observation that these mutations in PapG also led to a decrease in the levels of the adhesin incorporated into pilus tips (data not shown). Elimination of the last 14 residues from the MBP–PapG140 fusion protein abolished PapD binding, as expected. An MBP–lacZα fusion construct also did not interact with PapD and served as a negative control. These results are consistent with the crystallographic data from the PapD–PapG-peptide complex, as well as with NMR data on the binding of PapD to an 8mer PapG C-terminal peptide and with studies using peptides as inhibitors of complex formation between PapD and MBP–PapG140 (Karlsson et al., 1998). It should be noted that it is possible that the non-conserved hydrophilic residues may also play an important role in pilus assembly. It might even be that one side of the strand interacts with the chaperone and the other side is involved in the assembly of the pilus, but the exact nature of these interactions awaits further analyses. Effect of C-terminal mutations on the incorporation of PapA into the growing pilus To investigate further the role of the C-terminus in mediating subunit–subunit contacts, we examined the effects of mutations within PapA, the major component of the pilus rod, with respect to pilus assembly. We selected G150 and Y162 as the targets for site-directed mutagenesis, as these two highly conserved positions flank the conserved C-terminal motif. Sites within this region (i.e. positions 151–161) were avoided intentionally because these would also affect PapA–PapD interactions (as deduced from the results with PapG), which would significantly cloud the interpretation of any observed changes with respect to pilus assembly. Each of the PapA mutants was expressed in trans with a ΔpapA (papHCDJKEFG) operon. Pili from each of the isogenic strains were purified via MgCl2 precipitation and examined by SDS–PAGE (Figure 3A, lanes 6–8). Cells expressing either wild-type PapA or G150A-PapA produced pili as detected via the MgCl2 purification procedure. In contrast, no pili could be isolated from cells expressing the G150T- or Y162L-PapA variants, indicating that these mutations abolished the ability of PapA to be assembled into rods. Figure 3.(A) Crude extracts of surface tips/pili from strains expressing a papA gene encoding a C-terminal mutant in trans with a papHCDJKEFG operon were prepared and analyzed by SDS–PAGE (lanes 2–4). Pili could be purified via MgCl2-induced cross-linking of PapA rods from a strain expressing wild-type PapA, but not from strains expressing the G150T or Y162L mutants (lanes 6–8). Affinity purification of tips/pili expressed from these strains was conducted by applying samples to a slurry of Gal-α(1,4)-Gal–Sepharose beads. After extensive washing, the bound PapG-containing structures (i.e. tips and pili) were eluted specifically with ethyl-β-galabioside. Examination of these samples by SDS–PAGE revealed that some PapA molecules were in fact incorporated into the growing organelles (lanes 10–12). Lanes 5, 9 and 13 contain samples prepared from an isogenic strain lacking papA. The PapA subunits were visualized by either Coomassie Blue staining (shown) or Western blotting with α−PapDA2 antiserum. (B) Examination of the Gal-α(1,4)-Gal-purified structures by high-resolution EM revealed full-length pilus rods in the cases of wild-type PapA (panel 1), but only tip fibrillae in the cases of the G150T and Y162L mutants and the PapA deletion construct (panels 2, 3 and 4). Interestingly, small ‘knobs’ could be observed at the ends of many of the G150T- and Y162L-derived tips (not seen in the negative control), perhaps representing a partial helical turn of one or more PapA molecules. Download figure Download PowerPoint Since stable PapD–PapA complexes can be isolated from strains expressing these latter two variants (Bullitt et al., 1996), the absence of rods must have arisen as a consequence of either: (i) the inability of any PapA molecules to become incorporated into the growing organelle following the assembly of the tip fibrillum; or (ii) the inability of additional PapA subunits to assemble into a pilus rod subsequent to the incorporation of the first (or the first few) PapA subunit attached to the base of the tip fibrillum. In order to distinguish between these two possibilities, adhesive organelles expressed from these strains were purified via Gal-α(1,4)-Gal affinity chromatography and examined by both SDS–PAGE and high-resolution electron microscopy. This purification scheme relies on the tip-located PapG to bind to the receptor. In the case of wild-type pili, the tip fibrillum is joined to the adhesin and the rod is joined to the tip fibrillum, so the entire composite organelle was purified by this procedure as verified by quick-freeze deep-etch electron microscopy (Figure 3B, panel 1). Analysis of this material by SDS–PAGE revealed the presence of the PapA protein (Figure 3A, lanes 2, 6 and 10). In the absence of PapA, only tip fibrillae were purified (Figure 3B, panel 4) and analysis by SDS–PAGE revealed the lack of a PapA band (Figure 3A, lanes 5, 9 and 13). In the cases of the G150T and Y162L variants, only tip fibrillar structures were purified (Figure 3B, panels 2 and 3). However, examination of this material by SDS–PAGE revealed that the fibrillar structures contained PapA (Figure 3A, lanes 3–4, 7–8 and 11–12). Interestingly, small ‘knobs’ could be observed at the ends of many of the G150T- and Y162L-derived tips (not seen in the negative control), perhaps representing a partial helical turn of one or more of the incorporated PapA molecules. Structure of PapD–PapK-peptide complex In order to investigate the extent to which PapD caps the PapA C-terminal assembly surface, we attempted to co-crystallize PapD with a 19mer C-terminal PapA peptide, but solubility problems with the peptide hampered these attempts. However, we were successful in co-crystallizing PapD with a 19mer peptide corresponding to the C-terminal region of PapK, which possesses the same side chains within the conserved alternating hydrophobic motif as PapA (Tyr2′, Leu4′, Phe6′ and Ala8′, where the number denotes the residue's position relative to the C-terminus). The crystal structure of the PapD–PapK-peptide complex was solved to 2.8 Å resolution (Figure 4). Crystal data and refinement parameters are summarized in Table II. The peptide bound in an extended conformation along the outer G1 β-strand of PapD via a β-zipper motif comprised of seven main chain hydrogen bonds between the peptide and the G1 β-strand; this interaction effectively extended the β-sheet formed by PapD's G1, F1, C1 and D1 β-strands out to a fifth strand. The C-terminus of the peptide was anchored in the interdomain cleft of PapD by hydrogen bonds to the side chains of Arg8 and Lys112. Figure 4.Ribbon representation of the PapD–PapK-peptide crystal structure showing the peptide bound in an extended conformation along the G1 β-strand of PapD, effectively extending the β-sheet defined by strands G1, F1, C1 and D1 of PapD. The peptide is bound via a β-zipper motif comprised of main chain hydrogen bonds. The C-terminus of the peptide is anchored in the interdomain cleft by additional hydrogen bonds to Arg8 and Lys112 of PapD. A translucent β-strand is drawn through the K-peptide. The figure was generated using MOLSCRIPT (Kraulis, 1991) and Raster3D (Bacon and Anderson, 1988; Merritt and Murphy, 1994). Download figure Download PowerPoint Table 2. Crystal data and refinement parameters Space group C2221 Unit cell (Å) a 57.20 b 153.77 c 135.86 α = β = γ 90° Resolution (Å) 2.8 Percent complete (20–2.8 Å) 95.5 Percent complete (2.9–2.8 Å) 85.1 Protein atoms 3428 Ligand atoms 232 Total reflections 16 071 R.m.s.d. bond (Å) 0.019 R.m.s.d. angle 3.87° Rsyma 0.075 Rcrystb 0.192 a Rsym = ΣhΣi|Ihi − |/ΣhΣi Ihi, where Ihi and Ih are the intensities of the individual and mean structure factors, respectively. b Rcryst = Σ|Fobs − Fcalc|/ΣFobs, where Fobs and Fcalc are observed and calculated structure factor amplitudes, respectively. Only data with Fobs/σ(Fobs) >2.0 were used in the refinement. The Arg1′ side chain of the peptide made several van der Waals contacts with residues from domain 2 of PapD: Ile154, Thr170 and Ile194. All other contacts between the peptide and PapD involved residues from domain 1 of the chaperone. The side chain of the highly conserved Tyr2′ made limited van der Waals contact with the shallow pocket formed by PapD residues Leu4, Thr7, Thr109 and Ile111; interestingly, the hydroxyl group of the peptide's Tyr2′ side chain did not appear to hydrogen-bond with any residues of PapD. This suggests that while the conserved aromatic moiety might be important for chaperone binding, as deduced from the ELISA results above, the hydroxyl group might play a role in mediating subunit–subunit interactions. Indeed, substitution of Phe for the penultimate Tyr in PapA has been shown to alter the helical symmetry of the pilus rod (Bullitt et al., 1996). Additional van der Waals contacts with PapD were made by the side chains of Leu4′ and Ala8′, which are part of the conserved hydrophobic motif common to all pilus subunits; these made contact with the side chains of Ile105 and Leu107 from the conserved G1 β-strand of PapD (Figure 5). Figure 5.Ribbon representation of the PapD–PapK-peptide crystal structure, shown here with PapD's solvent-accessible connolly surface. The hydrophobic bed comprised of residues along PapD's G1 β-strand is highlighted in yellow. The inset provides a magnified view of the PapD–PapK-peptide contact interface. Note how the conserved alternating hydrophobic residues of the peptide interdigitate with the residues along PapD's G1 β-strand. The figure was generated using the Insight II modeling package (MSI, San Diego, CA). Download figure Download PowerPoint The first five N-terminal residues of the peptide displayed no electron density, and must therefore have been disordered in the crystal structure. Overall, these observations are in general agreement with the interactions seen in the previously determined crystal structure of the PapD–PapG-peptide complex, although the peptide side chains at positions 2′ and 6′ appear to make notably less extensive contacts with the chaperone. Remarkably, superimposition of the ligands from the PapD–PapG- and PapD–PapK-peptide crystal structures revealed that they adopt a virtually identical amide backbone conformation in the region that interacts with the G1 β-strand of PapD, despite the fact that side chain identities differ at 15 out of the 19 positions. This underscores the importance of the hydrogen bonding interactions comprising the β-zipper motif. Additional insight was gained through a comparison of the PapD structures from these two complexes as well as from the apo-PapD crystal structure (Holmgren and Brändén, 1989). The complete PapD Cα traces from all three crystal structures, as well as sectional Cα traces from selected regions of PapD, were superimposed, and positional root-mean-square deviations (r.m.s.ds) for corresponding atoms were calculated (Table III). This analysis revealed that only modest conformational changes occur upon peptide binding, except within the loop region connecting the F1 and G1 β-strands (Figure 6; Table IV). Here, a substantial degree of ordering occurs upon ligand binding that extends the apparent length of the G1 β-strand as defined by the corresponding Φ and Ψ torsion angles of the polypeptide backbone within this region. It was previously unknown if the changes in conformation of the F1–G1 loop seen in the PapD–PapG-peptide crystal structure were a result of ligand binding or only a consequence of crystal packing. The similar conformational changes seen in the PapD–PapK-peptide structure strongly argue that the reorganization of this region and the resulting elongation of the G1 β-strand are due in fact to peptide binding. Figure 6.(A) Superimposition of the Cα traces from the unbound and peptide-bound forms of PapD. (B) Superimposition of the Cα traces from domain 1 of the unbound and peptide-bound forms of PapD. Examination of the superimposed molecules revealed only modest conformational changes upon peptide binding, except within the F1–G1 mobile loop (Table IV). PapD residues 102–105 shift from their positions in the unbound form so as to extend the apparent length of the G1 strand upon binding of the peptides. (For this figure, the Cα traces of residues 1–80 from domain 1 were used to align the structures.) (C) Close up of the boxed region in (B). Residue numbers are indicated next to the appropriate Cα. The shifts in a given Cα's position upon ligand binding are given by the interatomic vectors. Download figure Download PowerPoint Table 3. Superimposition of PapD Cα traces from the apo-PapD structure and PapD–PapG/K-peptide complexes Molecules superimposed R.m.s.d. (Å) Entire moleculea (residues 1–218) Domain 1 (1–115) Domain 1b (1–80) Domain 1b (81–115) Domain 2 (125–218) PapDK on PapDG 0.927 0.948 0.429 1.374 0.557 PapD on PapDK 1.603 2.026 0.391 2.718 0.530 PapD on PapDG 1.694 1.855 0.449 2.526 0.646 a See Figure 6A. b See Figure 6B and C. Table 4. Interatomic distances between residues in the F1–G1 loop r
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