Biology subjects

Fleishman, S. J.

Publications and source records attributed to Fleishman, S. J..

23 records · Page 2Linked to original sources

bioRxiv2021

Biomolecular recognition of the glycan neoantigen CA19-9 by distinct antibodies

Glycans decorate cell surface, secreted glycoproteins and glycolipids. Altered glycans are often found in cancers. Despite their high diagnostic and therapeutic potentials, glycans are polar and flexible molecules that are quite challenging for the development and design of high-affinity binding antibodies. To understand the mechanisms by which glycan neoantigens are specifically recognized by antibodies, we analyze the biomolecular recognition of a single tumor-associated carbohydrate antigen CA19-9 by two distinct antibodies using X-ray crystallography. Despite the plasticity of glycans and the very different antigen-binding surfaces presented by the antibodies, both structures reveal an essentially identical extended CA19-9 conformer, suggesting that this conformers stability selects the antibodies. Starting from the bound structure of one of the antibodies, we use the AbLIFT computational method to design a variant with seven core mutations that exhibited tenfold improved affinity for CA19-9. The results reveal strategies used by antibodies to specifically recognize glycan antigens and show how automated antibody-optimization methods may be used to enhance the clinical potential of existing antibodies.

molecular biology↗

bioRxiv2020

A computationally designed fluorescent biosensor for D-serine

Solute-binding proteins (SBPs) have evolved to balance the demands of ligand affinity, thermostability and conformational change to accomplish diverse functions in small molecule transport, sensing and chemotaxis. Although the ligand-induced conformational changes that occur in SBPs make them useful components in biosensors, they are challenging targets for protein engineering and design. Here we have engineered a D-alanine-specific SBP into a fluorescent biosensor with specificity for the signaling molecule D-serine (D-serFS). This was achieved through binding site and remote mutations that improved affinity (KD = 6.7 {+/-} 0.5 M), specificity (40-fold increase vs. glycine), thermostability (Tm = 79 {degrees}C) and dynamic range (~14%). This sensor allowed measurement of physiologically relevant changes in D-serine concentration using two-photon excitation fluorescence microscopy in rat brain hippocampal slices. This work illustrates the functional trade-offs between protein dynamics, ligand affinity and thermostability, and how these must be balanced to achieve desirable activities in the engineering of complex, dynamic proteins.

biochemistry↗

bioRxiv2020

Coronacept - a potent immunoadhesin against SARS-CoV-2

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Computational analysis of mammalian ACE2 orthologues suggests various residues at the interface with the viral receptor binding domain that could facilitate tighter interaction compared to the human-ACE2. Introducing several mutations to the human-ACE2 resulted with significantly augmented affinity to the viral spike complex. This modified human-ACE2 fused to an Fc portion of an antibody makes a potent immunoadhesin that effectively targets SARS-CoV-2.

bioengineering↗

bioRxiv2020

De novo designed receptor transmembrane domains enhance CAR-T cell cytotoxicity and attenuate cytokine release

De novo designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass -helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs (proCARs) stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.

synthetic biology↗

bioRxiv2019

A lipophilicity-based energy function for membrane-protein modelling and design

Membrane-protein design is an exciting and increasingly successful research area which has led to landmarks including the design of stable and accurate membrane-integral proteins based on coiled-coil motifs. Design of topologically more complex proteins, such as most receptors, channels, and transporters, however, demands an energy function that balances contributions from intra-protein contacts and protein-membrane interactions. Recent advances in water-soluble all-atom energy functions have increased the accuracy in structure-prediction benchmarks. The plasma membrane, however, imposes different physical constraints on protein solvation. To understand these constraints, we recently developed a high-throughput experimental screen, called dsT{beta}L, and inferred apparent insertion energies for each amino acid at dozens of positions across the bacterial plasma membrane. Here, we express these profiles as lipophilicity energy terms in Rosetta and demonstrate that the new energy function outperforms previous ones in modelling and design benchmarks. Rosetta ab initio simulations starting from an extended chain recapitulate two-thirds of the experimentally determined structures of membrane-spanning homo-oligomers with <2.5 [A] root-mean-square deviation within the top-predicted five models. Furthermore, in two sequence-design benchmarks, the energy function improves discrimination of stabilizing point mutations and recapitulates natural membrane-protein sequences of known structure, thereby recommending this new energy function for membrane-protein modelling and design.

biochemistry↗