Biology subjects

Markovic, I.

Publications and source records attributed to Markovic, I..

4 recordsLinked to original sources

bioRxiv2024

De novo design of high-affinity single-domain antibodies

Antibody-based therapeutics have become indispensable in modern medicine, but traditional methods of antibody discovery often present with limitations in developability, cross-reactivity, and ethical concerns. While deep learning and generative approaches have shown promise in the design of high affinity protein binders, de novo antibody design remains challenging. Here, we present EvolveX, a structure-based computational pipeline for designing antibody fragments. EvolveX utilizes ModelX and empirical force field FoldX to optimize complementarity- determining regions (CDRs) and TANGO for aggregation analysis. We demonstrate the ability of EvolveX to redesign a single-domain VHH antibody fragment targeting mouse Vsig4 to address two challenges: enhancing stability and affinity for the original target and redesigning it for high affinity to the human ortholog. The redesigned variants of VHH fragments specific to mouse Vsig4 showed improved physicochemical properties, while retaining binding affinities comparable to the original version. Notably, EvolveX improved the binding affinity of VHHs to human Vsig4 by over 1000-fold, transforming low-affinity binders into nanomolar-affinity molecules. Structural analyses by X-ray crystallography and NMR confirmed the accuracy of the designs, which display optimized interactions with the antigen. NGS and re-modelling analysis further demonstrated the efficiency of FoldX-based design pipeline. Collectively, our study highlights EvolveXs potential to overcome current limitations in antibody design, offering a powerful tool for the development of next-generation therapeutics with enhanced specificity, stability, and efficacy.

bioinformatics↗

bioRxiv2022

Structural basis for the mechanism and antagonism of receptor signaling mediated by Interleukin-9 (IL-9)

Interleukin-9 (IL-9) is the hallmark cytokine in Th9 immunity and is also central to Innate Lymphocyte 2 (ILC2) biology. Furthermore, receptor signaling mediated by IL-9 has been linked to inflammatory and autoimmune diseases, and cancer. Despite its functional pleiotropy, the structure-function landscape of IL-9 had remained enigmatic. Here, we show via a combination of X-ray crystallography and NMR that human IL-9 adopts a helical bundle fold with unprecedented structural features among helical cytokines, including five disulfide bridges. Binding of IL-9 to the interdomain junction of IL-9R results in marked structural changes on the opposite face of IL-9 that prime the binary complex for recruiting the common gamma chain ({gamma}c) for signaling. Surprisingly, this tripartite cytokine-receptor assembly displays a markedly lower affinity than the IL-9: IL-9R complex, which we trace to distinct features of IL-9R that might destabilize the ternary complex. Furthermore, we developed monoclonal antibodies that antagonize IL-9 activity by sterically competing for the binding footprint of IL-9R. Collectively, we here provide a structural and mechanistic blueprint to facilitate interrogation and modulation of pleiotropic signaling outputs of IL-9 in physiology and disease.

immunology↗

bioRxiv2022

Mechanism of receptor assembly via the pleiotropic adipokine Leptin

The adipokine Leptin activates its type I cytokine receptor (LEP-R) in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility, and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Here, we show that Leptin:LEP-R assemblies adopt an unprecedented structure within the type I cytokine receptor family featuring 3:3 stoichiometry. We validate Leptin-induced trimerization of LEP-R in the plasma membrane of living cells via multicolor single molecule microscopy. In mediating such assemblies Leptin undergoes drastic restructuring that activates its site III for binding to the Ig-domain of an adjacent LEP-R molecule in the complex. These interactions are abolished by pathological mutations linked to obesity. Collectively, our study uncovers an evolutionarily conserved Leptin:LEP-R assembly as a new mechanistic blueprint for Leptin-mediated signaling in physiology and disease, including insights into how the lowly abundant signaling-competent isoforms of LEP-R can productively participate in signaling.

molecular biology↗

bioRxiv2021

Robust de novo design of protein binding proteins from target structural information alone

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains an outstanding challenge. We describe a general solution to this problem which starts with a broad exploration of the very large space of possible binding modes and interactions, and then intensifies the search in the most promising regions. We demonstrate its very broad applicability by de novo design of binding proteins to 12 diverse protein targets with very different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of four of the binder-target complexes, and all four are very close to the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvement of both. Our approach now enables targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

synthetic biology↗