Biology subjects

Fleishman, S.

Publications and source records attributed to Fleishman, S..

5 recordsLinked to original sources

bioRxiv2026

Structural and biochemical insights into HCV envelope proteins for germline-targeting vaccines

Despite effective antivirals, Hepatitis C remains a global health burden, reflecting the difficulty of eliciting broadly neutralizing antibodies (bnAbs). For the Hepatitis C virus, bnAb responses are biased toward VH1-69-encoded Abs targeting a conserved epitope on the E2 glycoprotein, suggesting that effective immunogens must engage the corresponding germline B cell receptors. However, the structural basis of germline recognition remains unclear. Here, we show that germline-reverted VH1-69 bnAbs recognize the E2 core domain in a manner closely resembling mature Abs binding. High-resolution structures reveal a conserved mode of engagement, indicating that key epitope features are accessible prior to affinity maturation. Guided by these insights, we engineered E2 variants to enhance germline Ab interactions and stability. Although these designs increased affinity, they did not improve engagement of naive B cells, highlighting a key constraint in germline-targeting strategies. Together, our findings define principles governing germline recognition and inform the rational design of an HCV vaccine.

biochemistry↗

bioRxiv2025

Optimizing stability in dynamic small-molecule binding proteins

The function of dynamic proteins is determined by the stability of distinct conformational states and the energy barriers that separate these states. For most dynamic proteins, the molecular details of the energy barriers are not known, implying a fundamental limit to the ability of protein design methods to engineer beneficial mutations without disrupting activity. We hypothesized that designing mutations that are compatible with structurally distinct equilibrium conformations may enable reliable stability design. We focus on periplasmic binding proteins (PBPs), a superfamily of dynamic proteins that change conformation from open to closed states in response to binding their small-molecule ligands. We find that the evolutionary constrained space of allowed mutations computed for one conformation is incompatible with the other. Therefore, putative conformational hinge points and interface residues were additionally constraint, and incompatible mutations were filtered out. Starting from four different PBPs, we designed a total of 16 stabilized variants with 7-28 mutations each. Our results show that design based on a single conformation with evolutionary constraints is not sufficient to maintain wild type-like binding affinity. Conversely, using a subset of mutations compatible with both conformations and structural constraints reliably enhances thermal stability while mitigating trade-offs in ligand binding. Our work demonstrates a straightforward method for the one-shot stabilization of dynamic proteins, which is critically required to generate robust starting points for thermostable and responsive biosensors.

biochemistry↗

bioRxiv2024

A computationally designed panel of diverse and selective peroxygenases for terpene oxyfunctionalization

Enzyme engineering has a critical role in the transition to economical, low-energy and environmentally friendly chemical production. Current approaches relying on costly iterations of mutation and selection are limited to reactions with a straightforward experimental readout and struggle to address mutational epistasis. We focus on unspecific peroxygenases (UPOs), prized engineering targets due to their ability to oxyfunctionalize diverse organic molecules of industrial and environmental value. To address the lack of scalable screening for UPO functions, we applied enzyme-design calculations to focus experiments. Starting from an AlphaFold2 model of MthUPO, the automated FuncLib algorithm generated 50 diverse active-site multipoint designs--all of which were functional. Screening against nine diverse terpenes revealed large improvements and new oxyfunctionalization products, resulting in molecules of high pharmaceutical and industrial value. We rationalized observed specificity changes using AI-based docking and molecular dynamics simulations, providing molecular insights that could generalize to engineering other UPOs. Thus, computational design and modeling can dramatically accelerate the urgently needed green transition of the chemical industry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/615329v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@3f8dceorg.highwire.dtl.DTLVardef@76ee48org.highwire.dtl.DTLVardef@15f2a7org.highwire.dtl.DTLVardef@edba31_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Computational design of BclxL inhibitors that target transmembrane domain interactions

Several methods have been developed to explore interactions among water-soluble proteins or regions of proteins. However, techniques to target transmembrane domains have not been examined thoroughly. Here we developed a novel computational approach to design transmembrane sequences that specifically modulate protein-protein interactions in the membrane. To illustrate this method we demonstrated that BclxL can interact with other members of the Bcl2 family through the transmembrane domain and that these interactions are necessary for BclxL control of cell death. Next, we designed sequences that specifically recognize and sequester the transmembrane domain of BclxL. Hence, we were able to prevent BclxL intra-membrane interactions and cancel its anti-apoptotic effect. These results advance our understanding of protein-protein interactions in membranes and provide new means to modulate them. Moreover, the success of our approach may trigger the development of a new generation of inhibitors targeting interactions between transmembrane domains.

biochemistry↗

bioRxiv2022

The C-terminal tail of CSNAP attenuates the CSN complex

Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitnation is the cullin-RING ligases (CRL). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumour progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises [~]300 complexes all of which are regulated by the COP9 signalosome complex (CSN). Therefore, the CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed within the CSN complex, in a groove formed by CSN3 and CSN8. We show that a 16 amino acid C-terminal peptide, derived from this CSN interacting region, can displace the endogenous CSNAP subunit from the complex. This, in turn, leads to a CSNAP null phenotype that attenuates CSN activity and consequently CRLs function. Overall, our findings emphasize the potential of a CSNAP-based peptide for CSN inhibition as a new therapeutic avenue.

biochemistry↗