Using a Low Denaturant Model To Explore the Conformational Features of Translocation-Active SecA

The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive post-translational secretion of preproteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, “closed” state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more “open”, has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain–domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1) and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon

Delicate Balance between Functionally Required Flexibility and Aggregation Risk in a β‑Rich Protein

Susceptibility to aggregation is general to proteins because of the potential for intermolecular interactions between hydrophobic stretches in their amino acid sequences. Protein aggregation has been implicated in several catastrophic diseases, yet we still lack in-depth understanding about how proteins are channeled to this state. Using a predominantly β-sheet protein whose folding has been explored in detail, cellular retinoic acid-binding protein 1 (CRABP1), as a model, we have tackled the challenge of understanding the links between a protein’s natural tendency to fold, ‘breathe’, and function with its propensity to misfold and aggregate. We identified near-native dynamic species that lead to aggregation and found that inherent structural fluctuations in the native protein, resulting in opening of the ligand-entry portal, expose hydrophobic residues on the most vulnerable aggregation-prone sequences in CRABP1. CRABP1 and related intracellullar lipid-binding proteins have not been reported to aggregate inside cells, and we speculate that the cellular concentration of their open, aggregation-prone conformations is sufficient for ligand binding but below the critical concentration for aggregation. Our finding provides an example of how nature fine-tunes a delicate balance between protein function, conformational variability, and aggregation vulnerability and implies that with the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable but controllable risk

DnaK residues identified from GNM-mobility play a key role in interdomain allostery.

<p>(<b>A</b>) GNM-predicted mobility profile, <<i>M_i</i>>|<i>₁₀</i>, evaluated for the ATP-bound structure <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Kityk1" target="_blank">[25]</a> (<b>D</b> in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g001" target="_blank"><b>Figure 1</b></a>), residues 4-530. The boxes on the upper abscissa show the residue ranges of subdomains IA, IB, IIA and IIB of the NBD, and the SBD (same color-code as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g001" target="_blank"><b>Figure 1</b></a>). Minima (T417, D481 and G506 in the hinge region) on SBD and linker residues (V389-L392) are marked with red dots. (<b>B</b>) Color-coded ribbon diagram based on mobility (<i>red</i>: most mobile; <i>blue</i>: least mobile). The most mobile region on the ATPase domain (colored <i>pink-red</i>) is the NEF-binding subdomain IIB (residues G228-V309). The global mobility is based on the <i>m</i> = 10 GNM lowest frequency modes, which account for 40% of the overall dynamics. Three highly mobile C-terminal residues are truncated to permit a clearer visualization. (<b>C</b>) ATP-induced fluorescence shift of T417A DnaK variant relative to that of WT DnaK, shown as first derivatives of fluorescence spectra recorded in the absence (solid) and presence (dashed) of 1 mM ATP. Data for WT DnaK are shown in black and those for T417A DnaK in red. The ATP-induced blue shift of the T417A DnaK variant (3.3±0.5 nm) indicates that it adopts a domain-docked conformation in the presence of ATP to an extent comparable to WT DnaK (blue shift 3.5±0.5 nm). (<b>D</b>) Since cell growth at elevated temperatures strictly depends upon functional DnaK, the ability to grow after heat shock was used to assess the <i>in vivo</i> function of the T417A DnaK variant. Plates are shown that have been incubated at 43 °C after inoculation by serial dilutions of DnaK^–<i>E. coli</i> cells transformed either with an empty vector or with plasmids encoding the WT or T417A DnaK variant. Despite its ability to undergo the normal ATP-induced conformational rearrangement, T417A DnaK cannot support growth after heat shock. (<b>E</b>) The ATPase rates of the T417A DnaK variant relative to WT rates: basal (grey) and upon stimulation by a model peptides (red: p5, CALLLSAPRR, and blue: NR, NRLLLTG). Note that the peptide-induced interdomain allosteric communication responsible for the ATPase stimulation is significantly reduced in T417A DnaK. (<b>F</b>) Growth of <i>E. coli</i> cells that lack SecB is stringently dependent on functional DnaK <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Smock2" target="_blank">[20]</a>. Shown are relative cell densities of cells transformed with either the empty vector or plasmids encoding IPTG-inducible DnaK genes for WT or T417A DnaK variant, either in the presence of arabinose (grey), glucose (red), and glucose + IPTG (blue). Arabinose and glucose induce or repress (respectively) the expression of the chromosomal copies of the <i>dnaK</i> and <i>dnaJ</i> genes, which have been placed under the control of the P_BAD promoter, while IPTG induces expression of the plasmid-encoded DnaK variants. The optical density to which these cultures grow correlates with the degree of <i>in-vivo</i> functionality of the expressed DnaK variants.</p

Results from coevolution analysis of Hsp70 family members.

<p>On panel <b>A</b>, the heat map based on PSICOV covariance predictions is displayed. The white rectangular frame encloses the portion corresponding to interdomain co-variances. Residue pairs distinguished by strongest interdomain signals are listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-t001" target="_blank"><b>Table 1</b></a> and illustrated in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g007" target="_blank"><b>Figure 7</b></a>. Those residues exhibiting high cumulative interdomain coevolutionary propensities are labeled and displayed in space-filling representation (labeled on panel <b>B</b>) and listed in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624.s007" target="_blank">Table S1</a></b>. The ribbon diagram is color-coded by the propensity of residues to exhibit coevolutionary patterns. NEF- and DnaJ-binding regions are highlighted. The DnaJ region is located mostly on the back of the area shown.</p

Emerging network of interactions establishing the communication between the DnaJ binding site (near E217 and V389) and the ATP-binding site of DnaK.

<p>(<b>A</b>) Two interconnected pathways, also coupled to each other (via E171-D194 interaction) are shown, belonging to the respective subdomains IA (<i>red</i>) and IIA (<i>green</i>) of the DnaK NBD. (<b>B</b>) Most on-pathway residues are conserved. L177, which plays a central role is distinguished by its coevolution with V389 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g008" target="_blank"><b>Figure 8</b></a>) and high influence/sensitivity with respect to the majority of displayed residues (<b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624.s006" target="_blank">Figure S6</a></b>).</p

L177 mediates interdomain communication via a cascade of interactions between highly co-evolving residues, including V389 at interdomain linker and T417 and the global hinge.

<p>(<b>A</b>–<b>B</b>) Highly coevolving residues shown in <i>orange spheres</i>, except for linker residues V389 and L392, colored <i>red</i>. Coevolving pairs of amino acids and their relative spatial positions, shown by stick representation, bridging between the global hinge (T417) and ATP-binding site (via A17). Inter-residue distances are in Å. MIp matrix portions corresponding to these coevolving pairs are indicated. (<i>bottom</i>) (<b>C</b>) ATP-induced fluorescence shift of DnaK variant L177A relative to that of WT DnaK, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g002" target="_blank">Figure 2c</a>. The ATP-induced blue shift of the L177A DnaK variant (2.0±0.5 nm) indicates that it is partially impaired in adopting a domain-docked conformation. (<b>D</b>) Growth-based functional assay at heat shock temperatures for L177A DnaK, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g002" target="_blank">Figure 2d</a>. Consistent with the partial impairment of L177A DnaK in domain docking, this DnaK variant cannot support growth after heat shock. (<b>E</b>) ATPase rates of L177A DnaK variant relative to WT rates, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g002" target="_blank"><b>Figure 2E</b></a>. Note that the peptide-induced interdomain allosteric communication responsible for the ATPase stimulation is significantly reduced in L177A DnaK. (<b>F</b>) Functional assay for L177A DnaK based on growth in the absence of SecB (as <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g002" target="_blank"><b>Figure 2F</b></a>), showing that this variant is severely impaired in <i>in-vivo</i> function relative to WT DnaK.</p

Close-up view of residue pairs distinguished by strong interdomain coevolutionary signals.

<p>Panels <b>A</b>–<b>C</b> display the structural position of residue pairs listed in the first part of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-t001" target="_blank"><b>Table 1</b></a>, which make tertiary contacts. Panel <b>D</b> displays the location of all listed residues on the structure.</p

Hsp70 allosteric cycle.

<p>In the ADP-bound state (<b>B</b>, <i>bottom-left</i>), the SBD (<i>purple</i>) and NBD are loosely connected by a flexible interdomain linker. NBD subdomains are colored <i>red</i> (subdomain IA; residues 3-38; 112-184), <i>blue</i> (IB; residues 39-111), <i>green</i> (IIA; residues 185-228; 310-388) and <i>orange</i> (IIB; residues 229-309) as indicated in the middle diagram. Upon replacement of ADP by ATP (both in <i>yellow</i>, <i>space-filling</i>) and ensuing release of substrate (<i>cyan, space-filling</i>), an open-SBD conformer is assumed (<b>D</b>, <i>upper-right)</i>, where the α-helical lid is docked onto the NBD, exposing and opening the substrate-binding site. The two domains allosterically regulate each other through intermediate allosterically active states illustrated in <b>A</b> and <b>C </b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Zhuravleva1" target="_blank">[8]</a>: ATP binding decreases the substrate-binding affinity of the SBD; substrate-binding increases the ATPase activity of the NBD. The co-chaperones, DnaJ and GrpE, assist in the hydrolysis and nucleotide exchange steps, respectively. Diagrams <b>B</b> and <b>D</b> were generated using the PDB files 1DKX <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Zhu1" target="_blank">[7]</a> and 4B9Q <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Kityk1" target="_blank">[25]</a>, respectively. <b>A</b> and <b>C</b> were generated manually—as their complete structure is unknown—based on the two mentioned structures and also on 1DKG <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Harrison1" target="_blank">[65]</a> and 2KHO <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Bertelsen1" target="_blank">[16]</a>.</p

Sensitivity profile of global hinge site, and network of conserved interactions between effector residues at subdomain IA and the linker.

<p>(<b>A</b>) Sensitivity profile of D481 (representing the hinge region), showing the predominance of effector residues belonging to subdomain IA helix 6 (D148, Q152, K155) and central residues (G6, I18, V139, R167) in the neighboring four strands, and those on β SBD. (<b>B</b>) Location of these effectors on DnaK structure. (<b>C</b>) Network of interactions between effectors in the neighborhood of D481 (<i>yellow stick</i>). Some inter-residue distances are shown as dashed lines; units in Å. ATP is shown in <i>yellow stick</i>, in the back. (<b>D</b>) Sequence logo plot describing the conservation level of these effectors. Symbol sizes scale with the frequency of different amino acid types at the sequence position.</p

Summary of computational results and relevance to experimental observations.

<p><i>(*) See also the distant pairs that exhibit strong coevolution signals, listed in </i><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-t001" target="_blank"><i>Table 1</i></a><i>, and labeled in </i><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g007" target="_blank"><i>Fig 7D</i></a><i>.</i></p