HAMP structure and function.

<p>(A) Side and top views of the dimeric HAMP four-helix bundle as observed in the trigonal crystal form. Core hydrophobic residues are shown in stick representation. (B) Crick angle deviation plot of the HAMP helices, as calculated with program samCC <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001776#pbio.1001776-DuninHorkawicz1" target="_blank">[52]</a>. (C) The ability of wild-type and mutant <i>cpxA</i> bacteria to respond to the periplasmic overproduction of either the wild-type MalE (open bars) or the folding-defective mutant MalE31 (light gray bars) and to the presence of 0.2% phenethyl alcohol (dark gray bars) was monitored by measuring β-galactosidase activity from a <i>cpxP</i>–<i>lacZ</i> fusion contained in the NS54 strain expressing the different <i>cpxA</i> alleles. The insert shows the cellular levels of CpxA analyzed by immunobloting membrane protein fractions prepared from the NS54 strain transformed by pLCB (lane 1), pLCBA_wt (lane 2), pLCBA₁₉₇ (lane 3), and pLCBA₂₂₈ (lane 4). The additional band observed in the immunoblot is a cross-reacting protein recognized by the antiserum that serves as a loading control. (D) Phosphotransferase activity of CpxA. Both full-length CpxA and CpxA_M228V proteins (10 µM) were first allowed to autophosphorylate for 20 min at 25°C in the presence of 1 mM ATP, and then an equimolar amount of CpxR_N (N-terminal receiver domain) was added to the reactions. Samples were removed at the indicated time points, and phospho-proteins were separated by Phos-tag acrylamide gel electrophoresis. (E) Autokinase activity of full-length CpxA and CpxA_M228V proteins as determined using radioactive ATP. It is worth noting that only a small fraction of the CpxA–Brij35 complex (∼0.1%) is phosphorylated at steady state, as estimated by PhosTag gels run under the same conditions used in the radioactive assays (unpublished data).</p

Identification of intragenic suppressors.

<p>A plasmid encoding CpxA_ΔP, a constitutive kinase CpxA variant lacking the sensor domain, was mutagenized and transformed into a <i>cpxA</i> null P_cpxP–<i>lacZ recA</i> strain background. The table shows the CpxA activity conferred by single mutant <i>cpxA</i> alleles in the NS54 strain. Point mutations were identified from colonies displaying a Lac⁻ phenotype in X-gal containing plates.</p

Conformational and dynamical asymmetry of the homodimer.

<p>(A) Superposition of all crystallographically independent CpxA_HDC dimers present in the five different crystal forms reported in this study. The main conformational difference between the distinct CpxA_HDC dimers consists in the position of one CA domain (left in the figure) relative to an invariant region containing both DHp domains and the second CA domain. (B) Phos-tag gel retardation autophosphorylation assay. The assay was performed with 10 µM CpxA_HDC and 1 mM ATP in 20 mM Hepes buffer (pH 7.6), 100 mM NaCl, 50 mM KCl, and 5 mM MgCl₂ at 25°C. At the indicated time points, 15 µl aliquots were removed and mixed with SDS loading buffer. Phospho-proteins were separated by Phos-tag acrylamide gel electrophoresis. (C) Total amount of CpxA_HDC-P and CpxA_HDC in each band was determined by densitometry analysis. The continuous lines were the best fits of the data to a single exponential term.</p

Overall structure of CpxA.

<p>(A) Linear representation of the prototypical CpxA domain organization. CpxA is an integral membrane receptor with a periplasmic sensor region (residues 29 to 163) flanked by two transmembrane helices (TM1 and TM2). TM2 connects the sensor domain to the cytosolic transmitter core (residues 188 to 457) formed by three domains: HAMP, DHp, and CA, rainbow colored from N-terminus–C-terminus (blue-red). (B) Cartoon representation of the CpxA_HDC homodimer in the trigonal crystal form. The homodimer is highly asymmetric due to helical kinks nearby Ser238 and Pro253 in helix α₂ (shown in stick representation) and large differences in the positioning and orientation of the two CA domains (shown in surface representation) with respect to the central DHp helical core.</p

Mechanical model for HK autophosphorylation control.

<p>The inactive kinase conformation (left) involves a putative symmetric conformation of CpxA [modeled from the structures of HK853 (PDB ID 2C2A) and wild-type Af1503 HAMP-EnvZ DHp chimera (PDB ID 3ZWR)], in which the two CA domains are sequestered in a nonproductive DHp–CA complex. The active kinase state (right, as observed in the trigonal crystal form) displays a highly asymmetric conformation of the HK homodimer. Propagated by conformational changes in the HAMP domain, the input signal induces a stress on the central DHp helices, promoting segmental helical motions that result in a strong dynamical asymmetry: one of the CA domains is highly mobile and can form a competent active site, whereas the second CA domain is retained in an inactive conformation by extended hydrophobic interactions with the DHp domain. The insert on the right shows these segmental helical movements (without the CA domains for clarity), in which each color represents a distinct rigid-body rotational movement. As a consequence of these movements, a gap broadens between two helices (indicated by a black arrow) and allows the partial exposure of core hydrophobic residues that contribute to sequester the second CA domain in an inactive conformation.</p

Autophosphorylating Michaelis complex.

<p>(A) Close view of the active site showing the residues directly involved in catalysis, as revealed by the hexagonal crystal structure of the CpxA_{HDC_M228V}–AMPPNP complex (a very similar active site architecture was observed for the trigonal and hexagonal crystal structures of wild-type CpxA_HDC in complex with ATP, obtained at lower resolution). The regions corresponding to the conserved N and H boxes are highlighted in dark green and yellow, respectively. (B) Activation of His248 for phosphoryl transfer. Hydrogen bonding interactions between the imidazole ring of His248, the adjacent acidic residue in the CA domain (Glu249) acting as a general base, and a polar residue from the DHp domain (Asn356) contribute to activate His248 for nucleophilic attack to γ-P of ATP. (C) Schematic representation of the CpxA homodimer illustrating the <i>trans</i>-autophosphorylation reaction. (D) Anchoring of Phe403 and Leu419 to the DHp four-helix bundle represented by its electrostatic surface. The DHp-sequestered (yellow) and mobile (green) CA domains are shown in cartoon representation.</p

Crystallographic data collection and refinement statistics.

<p>Values for the highest resolution shell are shown in parentheses.</p

Data collection and refinement statistics.

<p>Statistics for the highest-resolution shell are shown in parentheses.</p

Sequences and production of anti-glycosidase Affitins.

<p>(<b>A</b>) Secondary structure elements according to crystallographic structure are shown below the sequences. Residues that were randomized are labeled in red. Residues that are involved in interaction with less than 10% of the buried surface appear in bold letters. (<b>B</b>) Size-exclusion purification of CelD (blue) and Affitin H4 (red), E12 (gray) and H3 (black) using a Superdex75 16/60 column. Arrows show the molecular weight obtained at the defined retention volumes. (<b>C</b>) SDS-PAGE 15% showing the final purity of CelD and the Affitins. Molecular weights are indicated in kDa.</p

ITC analysis of anti-glycosidase binders.

<p>(<b>A</b>) ITC titrations at 25°C of Affitin H3 with CelD, Affitin E12 with CelD and Affitin H4 with HEWL. (<b>B</b>) Cross-recognitions were tested at 25°C for Affitin H3 with HEWL, Affitin E12 with HEWL and Affitin H4 with CelD. (<b>C</b>) ITC titrations at 60°C of Affitins H3 and E12 with CelD. The top panel for ITC shows data obtained from injections of Affitins while the bottom panel shows the integrated curve showing experimental points (filled squares) and the best fit (red line).</p