The Mitochondrial Protein Translocation Motor: Structural Conservation between the Human and Yeast Tim14/Pam18-Tim16/Pam16 co-Chaperones

Most of our knowledge regarding the process of protein import into mitochondria has come from research employing Saccharomyces cerevisiae as a model system. Recently, several mammalian homologues of the mitochondrial motor proteins were identified. Of particular interest for us is the human Tim14/Pam18-Tim16/Pam16 complex. We chose a structural approach in order to examine the evolutionary conservation between yeast Tim14/Pam18-Tim16/Pam16 proteins and their human homologues. For this purpose, we examined the structural properties of the purified human proteins and their interaction with their yeast homologues, in vitro. Our results show that the soluble domains of the human Tim14/Pam18 and Tim16/Pam16 proteins interact with their yeast counterparts, forming heterodimeric complexes and that these complexes interact with yeast mtHsp70

Mechanochemical basis of protein degradation by a double-ring AAA+ machine

Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ​ClpA enzyme from Escherichia coli, in complex with the ​ClpP peptidase, mechanically degrades proteins. We demonstrate that ​ClpA unfolds some protein substrates substantially faster than does the single-ring ​ClpX enzyme, which also degrades substrates in collaboration with ​ClpP. We find that ​ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ​ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Grant AI-16892

Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine

In the axial channels of ClpX and related hexameric AAA+ protein-remodeling rings, the pore-1 loops are thought to play important roles in engaging, mechanically unfolding, and translocating protein substrates. How these loops perform these functions and whether they also prevent substrate dissociation to ensure processive degradation by AAA+ proteases are open questions. Using ClpX pore-1-loop variants, single-molecule force spectroscopy, and ensemble assays, we find that the six pore-1 loops function synchronously to grip and unfold protein substrates during a power stroke but are not important in preventing substrate slipping between power strokes. The importance of grip strength is task dependent. ClpX variants with multiple mutant pore-1 loops translocate substrates as well as the wild-type enzyme against a resisting force but show unfolding defects and a higher frequency of substrate release. These problems are magnified for more mechanically stable target proteins, supporting a threshold model of substrate gripping

Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine

Hexameric ATP-dependent proteases and protein remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. Whether loops from multiple subunits act independently or coordinately in these processes is a critical aspect of the mechanism but is currently unknown for any AAA+ machine. By studying covalently linked hexamers of the Escherichia coli ​ClpX unfoldase bearing different numbers and configurations of wild-type and mutant pore loops, we show that loops function synergistically, and the number of wild-type loops required for efficient degradation is dependent on the stability of the protein substrate. Our results support a mechanism in which a power stroke initiated in one subunit of the ​ClpX hexamer results in the concurrent movement of all six pore loops, which coordinately grip and apply force to the substrate.National Institutes of Health (U.S.) (Grant GM-101988

The Pam18/Tim14–Pam16/Tim16 complex of the mitochondrial translocation motor: The formation of a stable complex from marginally stable proteins

The vast majority of mitochondrial proteins are imported from the cytosol. For matrix-localized proteins, the final step of translocation across the inner membrane is mediated by the mitochondrial translocation motor, of which mhsp70 is a key component. The ATP-dependent function of mhsp70 is regulated by a complex, composed of a J-protein (called Pam18 or Tim14) and a J-like protein (called Pam16 or Tim16), and the nucleotide exchange factor Mge1. In this study, we investigated the structural properties of a recombinant purified Pam18/Tim14–Pam16/Tim16 complex using cross-linking with the bifunctional reagent DSS and CD-spectroscopy. The results of the study show that both Pam18/Tim14 and Pam16/Tim16 are thermally unstable proteins that unfold at very low temperatures (Tm values of 16.5°C and 29°C, respectively). Upon mixing the proteins in vitro, or when both proteins are co-overexpressed in bacteria, Pam18/Tim14 and Pam16/Tim16 form a heterodimer that is thermally more stable than the individual proteins (Tm = 41°C). Analysis of the properties of the complex in GdnHCl shows that dissociation of the heterodimer is the limiting step in achieving full denaturation

High-resolution profiling of MHC II peptide presentation capacity reveals SARS-CoV-2 CD4 T cell targets and mechanisms of immune escape

Understanding peptide presentation by specific MHC alleles is fundamental for controlling physiological functions of T cells and harnessing them for therapeutic use. However, commonly used in silico predictions and mass spectroscopy have their limitations in precision, sensitivity, and throughput, particularly for MHC class II. Here, we present MEDi, a novel mammalian epitope display that allows an unbiased, affordable, high-resolution mapping of MHC peptide presentation capacity. Our platform provides a detailed picture by testing every antigen-derived peptide and is scalable to all the MHC II alleles. Given the urgent need to understand immune evasion for formulating effective responses to threats such as SARS-CoV-2, we provide a comprehensive analysis of the presentability of all SARS-CoV-2 peptides in the context of several HLA class II alleles. We show that several mutations arising in viral strains expanding globally resulted in reduced peptide presentability by multiple HLA class II alleles, while some increased it, suggesting alteration of MHC II presentation landscapes as a possible immune escape mechanism.ISSN:2375-254