2025 Hyper Recent •CC0 1.0 Universal

This work is dedicated to the public domain. No rights reserved.

Access Preprint From Server
July 2nd, 2025
Version: 2
The University of Kansas Medical Center
biochemistry
biorxiv

Dissecting the effects of single amino acid substitutions in SARS-CoV2 Mpro

Sreenivasan, S.Open in Google Scholar•Fontes, J.Open in Google Scholar•Swint-Kruse, L.Open in Google Scholar

Successfully predicting the effects of amino acid substitutions on protein function and stability remains challenging. Recent efforts to improve computational models have incluzded training and validation on high-throughput experimental datasets, such as those generated by deep mutational scanning (DMS) approaches. However, DMS signals typically conflate a substitution's effects on protein function with those on in vivo protein abundance; this limits the resolution of mechanistic insights that can be gleaned from DMS data. Distinguishing functional changes from abundance-related effects is particularly important for substitutions that exhibit intermediate outcomes (e.g., partial loss-of-function), which are difficult to predict. Here, we explored changes in in vivo abundance for substitutions at representative positions in the SARS-CoV-2 Main Protease (Mpro). For this study, we used previously published DMS results to identify "rheostat" positions, which are defined by having substitutions that sample a broad range of intermediate outcomes. We generated 10 substitutions at each of six positions and separately measured effects on function and abundance. Results revealed an ~45-fold range of change for abundance, demonstrating that it can make significant contributions to DMS outcomes. Moreover, the six tested positions showed diverse substitution sensitivities for function and abundance. Some positions influenced only one parameter. Others exhibited rheostatic effects on both parameters, which to our knowledge, provides the first example of such behavior. Since effects on function and abundance may arise through different biophysical bases, these results underscore the need for datasets that independently measure these parameters in order to build predictors with enhanced mechanistic insights.

Similar Papers

biorxiv
Thu Jul 03 2025
PCNA is a Nucleotide Exchange Factor for the Clamp Loader ATPase Complex
All life requires loading ring-shaped sliding clamp protein complexes onto DNA. The sliding clamp loader is a conserved AAA+ ATPase that binds the sliding clamp, opens the ring, and places it onto DNA. While recent structural work on both the canonical and \'alternative\' clamp loaders has shed light into how these machines perform their task once, it remains unclear how clamp loaders are recycled...
Pajak, J.
•
Landeck, J. T.
•
Liu, X.
•
Anand, K.
...•
Kelch, B. A.
biorxiv
Wed Jul 02 2025
Aggregation of HAPLN2, a component of the perinodal extracellular matrix, is a hallmark of physiological brain aging in mice
Protein aggregation is a hallmark of neurodegenerative diseases and is also observed in the brains of elderly individuals without such conditions, suggesting that aging drives the accumulation of protein aggregates. However, the comprehensive understanding of age-dependent protein aggregates involved in brain aging remains unclear. Here, we investigated proteins that become sarkosyl-insoluble with...
Watanabe, A.
•
Hirayama, S.
•
Kominato, I.
•
Marchese, S.
...•
Murata, S.
biorxiv
Wed Jul 02 2025
Structure and quenching of a bundle-shaped phycobilisome
Cyanobacteria use soluble antenna megacomplexes, phycobilisomes (PBS), to maximize light-harvesting efficiency and small photoswitchable orange carotenoid proteins (OCPs) to down-regulate PBS in high light. Among known PBS morphologies, the one from the basal cyanobacterial genus Gloeobacter still lacks detailed structural characterization. Here, we present the cryo-EM structure of the G. violaceu...
Burtseva, A. D.
•
Slonimskiy, Y. B.
•
Baymukhametov, T. N.
•
Sinetova, M. A.
...•
Sluchanko, N. N.
biorxiv
Wed Jul 02 2025
Urolithin B reduces the aggregate load of islet amyloid polypeptide in Caenorhabditis elegans
The progressive loss of pancreatic {beta}-cells is one of the defining features of Type 2 Diabetes Mellitus (T2DM), and is thought to be driven by the aggregation of islet amyloid polypeptide (IAPP). This highly amyloidogenic pancreatic hormone is co-secreted with insulin, and its elevated secretion can lead to toxic fibrillar aggregation. Despite numerous studies focusing on understanding the mol...
Akdag, M.
•
Ferreira, S.
•
Menezes, R.
•
Sinnige, T.
biorxiv
Wed Jul 02 2025
Structural basis for TORC2 activation
The Target of Rapamycin Complex 2 (TORC2) is a central node in signaling feedback loops serving to maintain biophysical homeostasis of the plasma membrane (PM). How TORC2 is regulated by mechanical perturbation of the PM is not well understood. To address this, we determined the cryo-electron microscopy structure of endogenous yeast TORC2 to up to 2.2 A resolution. Our model refines the position a...
Zou, L.
•
Tettamanti, M. G.
•
Bergmann, A.
•
Loewith, R.
•
Tafur, L.
biorxiv
Wed Jul 02 2025
Oxidant-mediated amino acid conversion of angiotensin II: Effects on gene expression in vascular smooth muscle cells.
Reactive oxygen species (ROS) play important roles in the pathogenesis of various diseases. One mechanism of protein oxidation is carbonylation, to which Arginine (Arg) and proline (Pro) are particularly susceptible. Both residues produce glutamic semialdehyde that can further be oxidized to glutamic acid (Glu). Thus, Arg, Pro, and Glu could be interchangeable, suggesting that ROS promote amino ac...
Suzuki, Y. J.
•
Teramoto, T.
biorxiv
Wed Jul 02 2025
DNA polymerase α-primase can function as a translesion DNA polymerase
Replication of cellular chromosomes requires a primase to generate short RNA primers to initiate genomic replication. While bacterial and archaeal primase generate short RNA primers, the eukaryotic primase, Pol alpha-primase, contains both RNA primase and DNA polymerase (pol) subunits that function together to form a >20 base hybrid RNA-DNA primer. Interestingly, the DNA Pol1 subunit of Pol alpha ...
Mayle, R.
•
Georgescu, R.
•
O'Donnell, M. E.
biorxiv
Wed Jul 02 2025
Sideroflexins enable mitochondrial transport of polar neutral amino acids
Mitochondria contribute to compartmentalized metabolism in eukaryotic cells, supporting key enzymatic reactions for cell function and energy homeostasis. However, this compartmentalization necessitates regulated metabolite transport across mitochondrial membranes. Although many transport proteins have been identified, several mitochondrial amino acid transporters remain largely uncharacterized. Us...
Block, S.
•
Chi, F.
•
Rosen, P. C.
•
Pineda, S. S.
...•
Vander Heiden, M. G.
biorxiv
Tue Jul 01 2025
Phase-Specific Antibiotic Resistance Mechanisms in an Escherichia coli B Strain
The majority of antibiotics developed to date target the fast-growing phase of bacteria, which typically occurs during active infection and resembles the exponential growth phase of laboratory-grown cultures. However, many pathogenic bacteria in the human body occupy environments where nutrients are limited and persist in a low-metabolic state, mirroring the stationary phase observed in laboratory...
Terrazas-Lopez, M.
•
Aitken, V.
•
Zeczycki, T. N.
•
Koculi, E.