RosettaCon 2022 posters#

Andrew Reckers | Predicting disruptions in protein conformational landscapes

While experimental and computational techniques to determine protein structure have improved dramatically in recent years, static structures do not provide a complete understanding of protein function. Mutations can cause allosteric conformational changes which are currently difficult to predict, often leading to aberrant function and disease. We are building a high-throughput pipeline for investigating mutation-driven conformational changes in proteins using a barcoded, computationally designed protein library analyzed with hydrogen-deuterium exchange with mass spectrometry (HDX/MS). Using the resulting dataset, we will develop a machine learning model that can predict the effects of mutations on the conformational states of proteins.

Neel Shanmugam | Protein Antivirals by Rapid Redesign of Tertiary Structures (PARROTS)

We propose a new, rapid and generalizable pipeline for engineering small and easy-to-produce alternatives to established antiviral protein therapeutics. PARROTS replaces native human serum albumin (HSA) helical bundles with computationally-designed helical bundles that tightly bind to viral antigens: HSA-traps.

Belen Sundberg | Reengineering allosteric regulation of bacterial PFK-1

Phosphofructokinase (PFK-1) catalyzes a rate-limiting step of glycolysis and is allosterically regulated by ligands whose binding can elicit conformational changes ~40Å away in the protein’s active sites. In cancer, allosteric regulation of PFK-1 by metabolites finetunes glycolytic flux to meet cellular demands. While the structure of PFK-1 is well conserved, eukaryotic orthologs are larger and bind more allosteric regulators than archaeal and bacterial PFK-1. Sequence comparison and biochemical mutation studies suggest that mammalian PFK-1 evolved by a process of tandem gene duplication and fusion, ultimately resulting in additional allosteric binding sites derived from duplicated catalytic and regulatory sites of ancestral PFK-1. One of these allosteric sites binds citrate, which allosterically inhibits PFK-1. Interestingly, cancer-associated mutations in the citrate binding site of the human platelet isoform of PFK-1, PFKP, are known to decrease PFKP inhibition by citrate, highlighting the importance of allosteric regulation in finetuning cancer metabolism. In order to elucidate the mechanisms behind the allosteric regulation of human PFK-1 by citrate, we sought to engineer its ancestral ortholog, bacterial PFK-1, to be allosterically inhibited by citrate binding using Rosetta protein design. Our most conservative computational design strategy models citrate in PFKP and grafts an identical citrate binding site in E. coli PFK-1, while our least conservative strategy builds a new citrate binding site in E. coli PFK-1. We will test our reengineered PFK-1 mutants for binding and inhibition by citrate and compare successful sequences with the living evolutionary record for PFK-1 to explore the evolution of allostery in this essential enzyme and its dysregulation in the context of cancer-associated mutations.