Schwarz, J. J. | bioRxiv Science

bioRxiv2023

The structural mechanism of MCIA complex assembly links mitochondrial redox pathways

The mitochondrial Complex I assembly (MCIA) complex is an essential player in the biogenesis of respiratory Complex I (CI), the multiprotein complex responsible for the initiation of oxidative phosphorylation (OXPHOS). It is not well understood how MCIA facilitates the assembly of CI. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, resulting in efflux of the FAD cofactor and redeployment of ACAD9 from fatty acid {beta}-oxidation (FAO) to CI assembly. We identify an adjacent -helix as a key structural element that specifically enables the CI assembly functionality of ACAD9, distinguishing it from its closely related VLCAD counterpart. Furthermore, we show that ECSIT is phosphorylated in vitro and ex cellulo and provide evidence that phosphorylation downregulates its association with ACAD9. Interestingly, ECSIT has previously been linked to the pathogenesis of Alzheimers disease and here we show that ECSIT phosphorylation in neuronal cells is reduced upon exposure to amyloid-{beta} (A{beta}) oligomers. These findings shed light on the assembly of the MCIA complex and implicate ECSIT as a potential reprogrammer of bioenergetic metabolic pathways that can be altered when mitochondria are affected by A{beta} toxicity, a hallmark of Alzheimers disease.

molecular biology↗

bioRxiv2021

Deamidation drives molecular aging of the SARS-CoV-2 spike receptor-binding motif

The spike is the main protein component of the SARS-CoV-2 virion surface. The spike receptor binding motif mediates recognition of the hACE2 receptor, a critical infection step, and is the preferential target for spike-neutralizing antibodies. Post-translational modifications of the spike receptor binding motif can modulate viral infectivity and immune response. We studied the spike protein in search for asparagine deamidation, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, affecting both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike. Similar deamidation hotspots are frequently found at the spike receptor-binding motifs of related sarbecoviruses, at positions that are mutated in emerging variants and in escape mutants from neutralizing antibodies. Asparagine residues 481 and 501 from the receptor-binding motif deamidate with a half-time of 16.5 and 123 days at 37 {degrees}C, respectively. This process is significantly slowed down at 4 {degrees}C, pointing at a strong dependence of spike molecular aging on the environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold. A model for deamidation of the full SARS-CoV-2 virion illustrates that deamidation of the spike receptor-binding motif leads to the accumulation in the virion surface of a chemically diverse spike population in a timescale of days. Our findings provide a mechanism for molecular aging of the spike, with significant consequences for understanding virus infectivity and vaccine development.

biochemistry↗