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

Representative DSC scans of WT Trx, pelB Trx and malE Trx.

<p>Scans were carried out in CGH-10 buffer (pH 7.4). The scan rate was 60°C /h and protein concentration was 0.2 mg/ml. Baseline subtracted excess heat capacity data as a function of temperature are shown. The data indicate that protein stability increases in the order malE Trx</p

CD spectra of WT Trx (−), pelB Trx (–) and malE Trx (···).(A) Far UV CD spectra of WT Trx (-), pelB Trx (–) and malE Trx (···) were obtained with 10 µM protein solution in CGH-10 buffer, pH 7.4 at 25°C with a 0.1 cm path-length cuvette.

<p>(B) Near UV CD spectra were obtained using protein concentrations of 600 µM, 400 µM and 250 µM for WT Trx, pelB Trx, and malE Trx respectively. Measurements were done in CGH-10 buffer, pH 7.4 at 25°C with a 0.2 cm path-length cuvette.</p

Insulin reduction assay for redox activity.

<p>Insulin aggregation following reduction was monitored by the increase in light scattering at 650 nm. Assay conditions were 0.1 M phosphate buffer, 2 mM EDTA, 0.13 mM porcine insulin, 0.33 mM DTT, and 5 µM protein. Protein identities are adjacent to each trace. Incubation mixture without protein served as negative control and WT Trx served as a positive control.</p

Effect of crowding agent (30% Ficoll) on Trx refolding.

<p>100 µM protein was denatured in 4 M GdmCl (CGH-10 buffer, pH 7.4), diluted and refolded into Ficoll containing buffer. Protein aggregation was monitored using the apparent absorbance at 320 nm. Bar graphs from left to right show data for proteins refolded at final protein concentrations of 2, 5, 7.5 and 10 µM each for WT Trx, pelB Trx and malE Trx respectively.</p

Aggregation propensity profiles of various signal peptides.

<p>Panels A–C show aggregation propensity profiles for pelB (empty circles,○) and malE (empty triangles,▵) sequences calculated using (A) Zyggregator, (B) PASTA, (C) AGGRESCAN. (D) Average hydrophobicity calculated using PREDBUR. Amino acid regions with Z_agg>1 are considered to be aggregation prone, whereas regions with Z_agg<0 were assumed to have low aggregation propensities. These upper and lower cut-offs are indicated by dashed line (–) and by dash-dot lines (–) respectively in panels A,E,F,G. The regions with aggregation propensity values above −0.02 are considered as hot-spots for aggregation by AGGRESCAN algorithm. The cut-off value is indicated by a dashed line (–) in C. Panel E-G show aggregation propensity profiles calculated using Zyggregator and average hydrophobicity calculated using PREDBUR for three previously studied soluble Trx fusion systems with phoA, treA, and pcoE signal sequences. Here, Z_agg is shown in filled circles (•), and average hydrophobicity is indicated with filled triangles (▴). The amino acid sequences for all the signal peptides are given in H. The locations of the AE mutation in pelB and malE are underlined.</p

Tricine-PAGE analysis of proteolytic digests of WT Trx, pelB Trx and malE Trx.

<p>These were performed at 37°C for 30 min and demonstrate that the signal peptides are protease accessible. Lanes 1–3 show undigested, papain and Proteinase K digested WT Trx. Lanes 4–6 show undigested, papain and Proteinase K digested pelB Trx. Lanes 7–9 show undigested, papain and Proteinase K digested malE Trx respectively. Proteolysis was stopped after 30 min by the addition of 1 µM Iodoacetic acid for Papain and 5 µM Phenylmethanesulfonic acid (PMSF) for Proteinase K. Samples were boiled with SDS-PAGE gel loading dye (2% SDS, 0.1% bromophenol blue, 10% Glycerol and 5% β-mercaptoethanol) prior to loading on the gel. Following electrophoresis, proteins were visualized by staining with Coomassie brilliant Blue R250. The relevant bands are enclosed by a box.</p

Thermal denaturation parameters at pH 7.4 for WT Trx, pelB Trx and malE Trx obtained from DSC.

<p>The protein concentration was 0.2 mg/ml, unless mentioned otherwise.</p>a<p>Since thermal unfolding is irreversible for this protein, these thermodynamic parameters are apparent values.</p><p>Enthalpies are calorimetric enthalpies.</p