Biology subjects

Smith, N. T.

Publications and source records attributed to Smith, N. T..

6 recordsLinked to original sources

bioRxiv2026

Massively scaled variant effect mapping across the MAPK pathway reveals the importance of molecular and cellular context

Variation alters inherent protein properties like thermodynamic stability and catalytic activity, and these intrinsic effects are shaped by the complex cellular environment. Signaling cascades typify this complexity, with tens or hundreds of proteins acting in concert through regulated states and interactions to transduce signals. Here, we measure the effect of 186,676 variants across 17 proteins in the RAS-MAPK pathway on protein abundance and pathway activity in human cells under diverse conditions, for a total of 503,099 variant effect measurements. We discovered 14,412 dominant negative variants across 12 proteins, nearly all previously unknown, that disrupt specific domains or interaction interfaces while leaving other functions intact and that are depleted from the human population. Measuring the abundance of 106,642 variants across 10 proteins under HSP90 inhibition showed that HSP90 shapes the effect of 67% of missense variants across the pathway, buffering destabilizing variants in both strong and weak clients. Our variant effect data, collected in the context of an intact proteome, enabled us to assess the functional importance of 395 experimentally and computationally modeled MAPK protein interactions. Using additional mutational scans, we resolved how the promiscuous switch II helix binding surface of KRAS accommodates overlapping interactions with LZTR1 and SOS1, revealing distinct allosteric networks that support each interaction. Thus, we establish a framework for measuring variant effects at unprecedented scale in human cells, illuminating fundamental MAPK pathway signaling mechanisms and demonstrating that cellular context profoundly shapes the effect of most variants.

genomics↗

bioRxiv2026

Evolution and remodeling of acceptor specificity in the NodZ α-1,6-fucosyltransferase

Glycosyltransferases have broad diversity in their acceptor substrates, yet the molecular and evolutionary basis of this diversity remains poorly understood. NodZ is an -1,6-fucosyltransferase that fucosylates Nod factors, lipochitooligosaccharide molecules involved in the establishment of rhizobium-legume symbioses. While NodZ is the only known bacterial member of the GT23 family of -1,6-fucosyltransferases, the determinants of its acceptor selectivity and the evolutionary origins thereof are unclear. Here, a phylogenetic analysis of 332 NodZ-like proteins revealed a distinct rhizobial clade and significantly greater structural diversity in the N-terminus than in the C-terminus. Kinetic analysis of a Mesorhizobium japonicum NodZ demonstrated selectivity for longer chitooligosaccharide acceptors (comprised of four to five GlcNAc units), while alanine scanning revealed W139 as a primary determinant of this chain length preference. We propose that W139 forms an aromatic platform that engages distal GlcNAc units present only in longer acceptors, coupling productive binding to acceptor chain length. We then used the NodZ phylogeny to reconstruct ancestral NodZ homolog sequences, which indicated that W139 emerged at the base of the rhizobial clade, and thus may represent a key evolutionary step towards canonical NodZ function. Additionally, the most recent resurrected ancestor was capable of fucosylating chitooligosaccharides with lower efficiency than the extant NodZ, suggesting that efficiency towards chitooligosaccharides was improved during the evolution of this family. Results described here demonstrate that evolutionary diversification of the N-terminal region can remodel acceptor specificity while preserving the conserved catalytic architecture of GT23 fucosyltransferases, providing a framework for understanding and engineering fucosyltransferase specificity.

biochemistry↗

bioRxiv2025

Characterization of bacterial fucokinase/GDP-fucose pyrophosphorylase (FKP) enzymes supports the evolution of interdomain communication and modularity in the FKP family

L-fucokinase (FUK) and GDP-fucose pyrophosphorylase (GFPP) salvage free L-fucose and synthesize the valuable nucleotide-sugar GDP-L-fucose (GDP-Fuc). Some organisms express these enzymes as one bifunctional polypeptide called L-fucokinase/GDP-fucose pyrophosphorylase (FKP), which has attracted attention for use in the chemoenzymatic synthesis of GDP-Fuc. Despite the documented use of the FKP from Bacteroides fragilis (BfFKP), the evolutionary origins of these enzymes and their relationships to monofunctional FUKs and GFPPs are poorly understood. We hypothesized that biochemical characterization of these proteins coupled with an evolutionary analysis would uncover the natural diversity of FKPs, facilitating the discovery of new biocatalysts. Phylogenetic and sequence similarity network (SSN) analyses distinguished FKPs from FUKs and GFPPs, suggesting that FKPs originate from one ancestral fusion event between these domains. To evaluate how environmental factors might select for functional diversity within the FKP family, we recombinantly expressed and purified a putative FKP from the thermophilic bacterium Thermophagus xiamenensis (TxFKP). This enzyme exhibited in vitro kinase and pyrophosphorylase activities and demonstrated subtle kinetic differences compared to BfFKP. While alanine scanning mutational analysis of the TxFKP FUK and GFPP domains supported the role of conserved residues that TxFKP uses to coordinate substrate binding and catalysis, other mutations in the TxFKP GFPP domain influenced kinase activity differentially for the substrates L-fucose and D-arabinose, showing an unprecedented role for the GFPP domain in FUK substrate specificity. Finally, thermal shift profiles of TxFKP and BfFKP were biphasic and provided new insights into how these enzymes have evolved to respond to different sugar substrates.

biochemistry↗

bioRxiv2024

Taxonomic distribution of SbmA/BacA and BacA-like antimicrobial peptide transporters suggests independent recruitment and convergent evolution in host-microbe interactions

Small, antimicrobial peptides are often produced by eukaryotes to control bacterial populations in both pathogenic and mutualistic symbioses. These include proline-rich mammalian immune peptides and cysteine-rich peptides produced by legume plants in symbiosis with rhizobia. The fitness of the bacterial partner is dependent upon their ability to persist in the presence of these antimicrobial peptides. In the case of Escherichia coli and Mycobacterium tuberculosis pathogens and nitrogen-fixing legume symbionts (rhizobia), the ability to survive exposure to these peptides depends on peptide transporters called SbmA (also known as BacA) or BclA (for BacA-like). However, how broadly these transporters are distributed amongst bacteria, and their evolutionary history, is poorly understood. Here, we used hidden Markov models, phylogenetic analysis, and sequence similarity networks to examine the distribution of SbmA/BacA and BclA proteins across a representative set of 1,255 species from across the domain Bacteria. We identified a total of 71 and 177 SbmA/BacA and BclA proteins, respectively. Phylogenetic and sequence similarity analyses suggest that these protein families likely did not evolve from a common ancestor and that their functional similarity is instead a result of convergent evolution. In vitro sensitivity assays using the legume peptide NCR247 and several of the newly-identified BclA proteins confirmed that transport of antimicrobial peptides is a common feature of this protein family. Analysis of the taxonomic distribution of these proteins showed that SbmA/BacA orthologs were encoded only by species in the phylum Pseudomonadota and that they were primarily identified in just two orders: Hyphomicrobiales (class Alphaproteobacteria) and Enterobacterales (class Gammaproteobacteria). BclA orthologs were somewhat more broadly distributed and were found in clusters across four phyla. These included several orders of the phyla Pseudomonadota and Cyanobacteriota, as well as the order Mycobacteriales (phylum Actinomycetota) and the class Negativicutes (phylum Bacillota). Notably, many of the clades enriched for species encoding BacA or BclA orthologs also include many species known to interact with eukaryotic hosts in mutualistic or pathogenic interactions. Collectively, these observations suggest that SbmA/BacA and BclA proteins have been repeatedly co-opted to facilitate both mutualistic and pathogenic associations with eukaryotic hosts by allowing bacteria to cope with host-encoded antimicrobial peptides.

microbiology↗

bioRxiv2023

A multiplex, prime editing framework for identifying drug resistance variants at scale

CRISPR-based genome editing has revolutionized functional genomics, enabling screens in which thousands of perturbations of either gene expression or primary genome sequence can be competitively assayed in single experiments. However, for libraries of specific mutations, a challenge of CRISPR-based screening methods such as saturation genome editing is that only one region (e.g. one exon) can be studied per experiment. Here we describe prime-SGE ("prime saturation genome editing"), a new framework based on prime editing, in which libraries of specific mutations can be installed into genes throughout the genome and functionally assessed in a single, multiplex experiment. Prime-SGE is based on quantifying the abundance of prime editing guide RNAs (pegRNAs) in the context of a functional selection, rather than quantifying the mutations themselves. We apply prime-SGE to assay thousands of single nucleotide changes in eight oncogenes for their ability to confer drug resistance to three EGFR tyrosine kinase inhibitors. Although currently restricted to positive selection screens by the limited efficiency of prime editing, our strategy opens the door to the possibility of functionally assaying vast numbers of precise mutations at locations throughout the genome.

genomics↗

bioRxiv2021

The Foster method: Rapid and non-invasive detection of clinically relevant American Foulbrood disease levels using eDNA sampling and a dual-target qPCR assay, with its potential for other hive pathogens.

Clinical signs of American Foulbrood (AFB) can be difficult to diagnose and thus disease is often missed and leads to further spread. Diagnosis is centred on the beekeepers skill in recognising clinical symptoms - a highly subjective and time-consuming activity. Previous testing methods have relied on sampling that necessitates dismantling the hive and/or requires multiple visits to retrieve passive samples. The Foster method is a novel environmental DNA sampling method using colony entrance swabs together with a dual-target qPCR for Paenibacillus larvae: the causative bacteria of AFB disease. The quantification data generated can be used to detect hives with clinically significant infections, even before visual symptoms are apparent. Such a sampling method will be applicable to other bee pathogens and incursion pests. ImportanceDiscovery of American foulbrood disease in a honeybee colony typically means the destruction of the bees and hive by burning, in New Zealand and many other countries. This discovery is typically by visual examination of capped brood by the beekeeper - a subjective skill that means the disease is being missed or not recognised. It is a time-consuming and exacting process to inspect hives for AFB. Here we present a novel rapid sampling method that does not require opening/ dismantling the hive, in conjunction with a dual-target quantitative PCR assay for the bacteria responsible, Paenibacillus larvae. Using the resulting quantitative data, hives presenting visual clinical symptoms or likely to soon become clinical, can be determined and the hives dealt with appropriately before further spread occurs. This study provides the basis for a novel way of sampling for honeybee pathogens and pests.

microbiology↗