Hydrophobically stabilized open state for the lateral gate of the Sec translocon

The Sec translocon is a central component of cellular pathways for protein translocation and membrane integration. Using both atomistic and coarse-grained molecular simulations, we investigate the conformational landscape of the translocon and explore the role of peptide substrates in the regulation of the translocation and integration pathways. Inclusion of a hydrophobic peptide substrate in the translocon stabilizes the opening of the lateral gate for membrane integration, whereas a hydrophilic peptide substrate favors the closed lateral gate conformation. The relative orientation of the plug moiety and a peptide substrate within the translocon channel is similarly dependent on whether the substrate is hydrophobic or hydrophilic in character, and the energetics of the translocon lateral gate opening in the presence of a peptide substrate is governed by the energetics of the peptide interface with the membrane. Implications of these results for the regulation of Sec-mediated pathways for protein translocation vs. membrane integration are discussed

SecA-dependent quality control of intracellular protein localization

Complex secretion machineries mediate protein translocation across cellular membranes. These machines typically recognize their substrates via signal sequences, which are required for proper targeting to the translocon. We report that during posttranslational secretion the widely conserved targeting factor SecA performs a quality-control function that is based on a general chaperone activity. This quality-control mechanism involves assisted folding of signal sequenceless proteins, thereby excluding them from the secretion process. These results suggest that SecA channels proteins into one of two key pathways, posttranslational secretion or folding in the cytoplasm. Implications of this finding for intracellular protein localization are discussed

Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase

In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane. SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation. Here we report the three-dimensional structure of the SecA protein of Mycobacterium tuberculosis. Each subunit of the homodimer contains a “motor” domain and a translocation domain. The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein. Knowledge of this structure provides a framework for further elucidation of the translocation process

Prediction of signal peptides in archaea

Computational prediction of signal peptides (SPs) and their cleavage sites is of great importance in computational biology; however, currently there is no available method capable of predicting reliably the SPs of archaea, due to the limited amount of experimentally verified proteins with SPs. We performed an extensive literature search in order to identify archaeal proteins having experimentally verified SP and managed to find 69 such proteins, the largest number ever reported. A detailed analysis of these sequences revealed some unique features of the SPs of archaea, such as the unique amino acid composition of the hydrophobic region with a higher than expected occurrence of isoleucine, and a cleavage site resembling more the sequences of gram-positives with almost equal amounts of alanine and valine at the position-3 before the cleavage site and a dominant alanine at position-1, followed in abundance by serine and glycine. Using these proteins as a training set, we trained a hidden Markov model method that predicts the presence of the SPs and their cleavage sites and also discriminates such proteins from cytoplasmic and transmembrane ones. The method performs satisfactorily, yielding a 35-fold cross-validation procedure, a sensitivity of 100% and specificity 98.41% with the Matthews' correlation coefficient being equal to 0.964. This particular method is currently the only available method for the prediction of secretory SPs in archaea, and performs consistently and significantly better compared with other available predictors that were trained on sequences of eukaryotic or bacterial origin. Searching 48 completely sequenced archaeal genomes we identified 9437 putative SPs. The method, PRED-SIGNAL, and the results are freely available for academic users at http://bioinformatics.biol.uoa.gr/PRED-SIGNAL/ and we anticipate that it will be a valuable tool for the computational analysis of archaeal genomes