Biology subjects

Leander, M.

Publications and source records attributed to Leander, M..

4 recordsLinked to original sources

bioRxiv2025

High-throughput discovery of transmembrane helix dimers from human single-pass membrane proteins with TOXGREEN sort-seq

The oligomerization of the transmembrane helices of single-pass membrane proteins is crucial to biological function and its misregulation can lead to many diseases. The study of transmembrane helix oligomerization is facilitated by the availability of genetic reporter assays, which are essential tools for understanding the organization and biology of single-pass systems. In particular, reporter assays are crucial for mapping the oligomerization interfaces of transmembrane helices through scanning mutagenesis but their application is limited by the need to clone and measure each construct individually. Here, we present "TOXGREEN sort-seq", a high-throughput version of the TOXGREEN assay that enables the direct measurement of transmembrane helix oligomerization in large libraries using fluorescence-activated cell sorting and next-generation sequencing. We show that TOXGREEN sort-seq is robust and reproduce the direct measurements of individual constructs with good accuracy and sensitivity. The method produced high-quality mutational profiles from a library of 17,400 constructs designed to probe the interface of 100 potential GASright dimers predicted from sequences of human single-pass membrane proteins. We report the validated structural model of twelve dimers involved in a variety of biological functions, including immune response (interleukin-22 receptor subunit alpha-1, butyrophilin-like protein 3, hepatitis A virus cellular receptor 2), transport (transferrin receptor protein 1), and cell-surface signaling and proliferation (syndecan-3; semaphorins 5A, 6B and 6D). Remarkably, all three semaphorins in the dataset formed strong dimers and produced mutational profiles consistent with the computational structure. These findings open the possibility that dimerization may be relevant to these proteins activity and provide a validated interface for assessing their biological role.

biophysics↗

bioRxiv2022

Deep mutational scanning and machine learning reveal structural and molecular rules governing allosteric hotspots in homologous proteins

A fundamental question in protein science is where allosteric hotspots - residues critical for allosteric signaling - are located, and what properties differentiate them. We carried out deep mutational scanning (DMS) of four homologous bacterial allosteric transcription factors (aTF) to identify hotspots and built a machine learning model with this data to glean the structural and molecular properties of allosteric hotspots. We found hotspots to be distributed protein-wide rather than being restricted to "pathways" linking allosteric and active sites as is commonly assumed. Despite structural homology, the location of hotspots was not superimposable across the aTFs. However, common signatures emerged when comparing hotspots coincident with long-range interactions, suggesting that the allosteric mechanism is conserved among the homologs despite differences in molecular details. Machine learning with our large DMS datasets revealed that global structural and dynamic properties to be a strong predictor of whether a residue is a hotspot than local and physicochemical properties. Furthermore, a model trained on one protein can predict hotspots in a homolog. In summary, the overall allosteric mechanism is embedded in the structural fold of the aTF family, but the finer, molecular details are sequence-specific.

biochemistry↗

bioRxiv2020

Mapping the Functional Landscape of the Receptor Binding Domain of T7 Bacteriophage by Deep Mutational Scanning

The interaction between a bacteriophage and its host is mediated by the phages receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, the molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP using a novel phage genome engineering method called ORACLE (Optimized Recombination, Accumulation and Library Expression). ORACLE is a high-throughput, locus-specific, sequence-programmable method to create a large, unbiased library of phage variants at a targeted gene locus. Using ORACLE, we generated all single amino acid substitutions at every site (1660 variants) of the tip domain to quantify the functional role of all variants on multiple bacterial hosts. This rich dataset allowed us to cross compare functional profiles of each host to precisely identify regions of functional importance, many which were previously unknown. Host-specific substitution patterns displayed differences in site specificity and physicochemical properties of mutations indicating exquisite adaptation to individual hosts. Comparison of enriched variants across hosts also revealed a tradeoff between activity and host range. We discovered gain-of-function variants effective against resistant hosts and host-constricting variants that selectively eliminated certain hosts. We demonstrate therapeutic utility against uropathogenic E. coli by engineering a highly active T7 variants to avert emergence of spontaneous resistance of the pathogen. Our approach presents a generalized framework for systematic and comprehensive characterization of sequence-function relationships in phages on an unprecedented scale.

synthetic biology↗

bioRxiv2020

Functional Plasticity and Evolutionary Adaptation of Allosteric Regulation

Allostery is a fundamental regulatory mechanism of protein function. Despite notable advances, understanding the molecular determinants of allostery remains an elusive goal. Our current knowledge of allostery is principally shaped by a structure-centric view which makes it difficult to understand the decentralized character of allostery. We present a function-centric approach using deep mutational scanning to elucidate the molecular basis and underlying functional landscape of allostery. We show that allosteric signaling exhibits a high-degree of functional plasticity and redundancy through myriad mutational pathways. Residues critical for allosteric signaling are surprisingly poorly conserved while those required for structural integrity are highly conserved, suggesting evolutionary pressure to preserve fold over function. Our results suggest multiple solutions to the thermodynamic conditions of cooperativity, in contrast to the common view of a finely-tuned allosteric residue network maintained under selection.

biochemistry↗