Swapna, G. | Search bioRxiv

bioRxiv2024

Sifting Through the Noise: A Computational Pipeline for Accurate Prioritization of Protein- Protein Binding Candidates in High-Throughput Protein Libraries

Identifying the interactome for a protein of interest is challenging due to the large number of possible binders. High-throughput experimental approaches narrow down possible binding partners, but often include false positives. Furthermore, they provide no information about what the binding region is (e.g. the binding epitope). We introduce a novel computational pipeline based on an AlphaFold2 (AF) Competition Assay (AF-CBA) to identify proteins that bind a target of interest from a pull-down experiment, along with the binding epitope. Our focus is on proteins that bind the Extraterminal (ET) domain of Bromo and Extraterminal domain (BET) proteins, but we also introduce nine additional systems to show transferability to other peptide-protein systems. We describe a series of limitations to the methodology based on intrinsic deficiencies to AF and AF-CBA, to help users identify scenarios where the approach will be most useful. Given the speed and accuracy of the methodology, we expect it to be generally applicable to facilitate target selection for experimental verification starting from high-throughput protein libraries. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/576374v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b90071org.highwire.dtl.DTLVardef@1cc2f66org.highwire.dtl.DTLVardef@3c1109org.highwire.dtl.DTLVardef@1827353_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

The broad host range bacteriophage Mu employs a novel methylcarbamoyl modification to protect its DNA from diverse host restriction systems. Biosynthesis of the unusual modification is a longstanding mystery. Moreover, isolation of Mom, the phage protein involved in the modification has remained elusive to date. Here, we characterized the co-factor and metal binding properties of Mom and provide a molecular mechanism to explain methylcarbamoylation by Mom. Our computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom, predicting acetyl CoA as its co-factor. We demonstrate that Mom binds to acetyl CoA and identify the active site. Puzzlingly, none of the > 309,000 GNAT members identified so far catalyze Mom-like modification of their respective substrates. Besides, conventional acid-base catalysis deployed by typical acetyltransferases cannot support methylcarbamoylation of adenine seen in Mu phage. In contrast, free radical-chemistry, catalyzed by Fe-S cluster or transition metal ions can explain the seemingly challenging reaction between acetyl CoA and DNA. We discovered that Mom is an iron-binding protein, with the Fe2+/3+ ion colocalized with acetyl CoA in the active site of Mom. Mutants defective for binding Fe2+/3+ or acetyl CoA demonstrated compromised activity, indicating their importance in the DNA modification reaction. Iron-binding in the GNAT active site is unprecedented and represents a small step in the evolution of Mom from the ancestral acetyltransferase fold. Yet, the tiny step allows a giant chemical leap from usual acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture. SummaryStudying the arms race between bacteria and their viruses (bacteriophages or phages) is key to understanding microbial life and its complexity. An unprecedented DNA modification shields phage Mu from bacterial restriction endonucleases that destroy incoming phage DNA. Nothing is known of how the modification is brought about, except that a phage protein Mom is involved. Here, we discover acetyl CoA and iron as key requirements for the modification. We explain how by evolving the ability to bind iron - a transition metal capable of generating highly reactive free radicals, a well-studied scaffold like the acetyltransferase fold can gain novel catalytic prowess in Mom. These findings have broad implications for gene editing technologies and therapeutic application of phages.

biochemistry↗