HCLS1-associated protein X-1 (HAX1) is a multifunctional mitochondrial protein involved in the regulation of apoptosis, a crucial process of programmed cell death, and mRNA processing. Despite its significance, limited structural data is available for HAX1, hindering a comprehensive understanding of its biological function. Notably, the caseinolytic mitochondrial matrix peptidase chaperone subunit B (CLPB) has been identified as an interacting partner of HAX1, yet the biophysical properties and binding affinity governing their interaction remain poorly defined. In this study, we present a thorough biophysical characterization of full-length human HAX1 and CLPB, accomplished through recombinant expression and purification. By employing size exclusion chromatography, dynamic light scattering, and circular dichroism spectroscopy, we successfully established their biophysical properties, revealing contrasting structural features, with CLPB displaying α-helical content and HAX1 exhibiting a disordered nature. Moreover, we employed solution-state nuclear magnetic resonance (NMR) spectroscopy to probe their binding affinity. Our findings demonstrate the formation of stable multimeric complexes between HAX1 and CLPB, and we quantified a dissociation constant in the low range of micro-molar for their high affinity interaction. These results lay the foundation for further in-depth investigations into the dynamics and energetics governing the HAX1-CLPB interaction, ultimately contributing to a comprehensive understanding of their functional mechanisms.

The mitochondrial pyruvate carrier (Mpc) plays an indispensable role in the transport of pyruvates across the mitochondrial inner membrane. Despite the two distinct homologous proteins, Mpc1 and Mpc2, were identified in 2012, there are still controversies on the basic functional units and oligomeric state of Mpc complexes. In this study, yeast Mpc1 and Mpc2 proteins were expressed in a prokaryotic heterologous system. Both homo- and hetero-dimers were successfully reconstituted in mixed detergents. Interactions among Mpc monomers were recorded utilizing paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) methods. By single-channel patch-clamp assays, we discovered that both the Mpc1–Mpc2 hetero-dimer and Mpc1 homo-dimer are able to transport K⁺ ions. Furthermore, the Mpc1–Mpc2 hetero-dimer demonstrated the ability to transport pyruvates, at a rate significantly higher than that of the Mpc1 homo-dimer, indicating that it could be the basic functional unit of Mpc complexes. Our findings provide valuable insights for further structural determination and the study of the transport mechanism of Mpc complexes.

In mitochondria, the major subunits of oxidative phosphorylation complexes are translated by the mitochondrial ribosome (mito-ribosome). The correct insertion and assembly of these subunits into the inner mitochondrial membrane (IMM) are facilitated by mitochondrial oxidase assembly protein 1 (Oxa1) during the translation process. This co-translational insertion process involves an association between the mito-ribosome and the C-terminus of Oxa1 (Oxa1-CTD) Nuclear magnetic resonance (NMR) methods were mainly used to investigate the structural characterization of yeast Oxa1-CTD and its mode of interaction with the E. coli 70S ribosome. Oxa1-CTD forms a transient α-helical structure within the residues P342–Q385, which were reported to form an α-helix when combining with the ribosome. Two conserved contact sites that could interact with the ribosome were further identified. The first site was located on the very end of the N-terminus (V321–I327), and the second one encompassed a stretch of amino acid residues I348–Q370. Based on our discoveries and previous reports, a model has been proposed in which Oxa1-CTD interacts with ribosomes, accompanied by transient-to-stable transitions at the second contact site. These observations may enhance our understanding of the potential role of Oxa1-CTD in facilitating the assembly of oxidative phosphorylation complexes and provide insight into the structural characteristics of Oxa1-CTD.