Biology subjects

Orban, J.

Publications and source records attributed to Orban, J..

10 recordsLinked to original sources

bioRxiv2026

A Conserved Flexible N-Terminal Domain Tunes the Calcium Sensitivity of Sorcin by Stabilizing Its Active Conformation

Sorcin is a penta-EF-hand Ca2+-binding protein that acts as a Ca2+ sensor and regulator of Ca2+ homeostasis. Although the structure and Ca2+-dependent activation of Sorcin is well characterized, the function of its flexible N-terminal domain (NTD) remains unclear. We combined sequence analysis, Ca2+-induced aggregation assays, multidimensional NMR spectroscopy, and long-timescale molecular dynamics (MD) simulations to define the NTDs role in Sorcin activation. Sequence comparisons showed that the NTD is conserved across vertebrates despite its intrinsic disorder, indicating functional importance. NTD deletion markedly reduced Ca2+ responsiveness. Relative to full-length Sorcin, the construct with NTD truncation required more than twofold higher Ca2+ concentrations and over tenfold higher protein concentrations to initiate aggregation, while aggregation kinetics slowed by nearly three orders of magnitude. Temperature-dependent measurements yielded an apparent activation energy of 36.7 kJ{middle dot}mol-1, consistent with aggregation driven by Ca2+-induced conformational activation rather than denaturation. NMR chemical shift perturbations localized the effects of NTD removal to the EF-hand Ca2+-binding loops and adjacent D-helix. Apo-state MD simulations revealed transient intra- and intermolecular NTD-SCBD contacts that explain some perturbations and support direct and allosteric regulation. Ca2+-bound simulations further showed that SCBD departs more readily from the crystallographic active conformation than full-length Sorcin, indicating that the NTD stabilizes the active state through dynamic contacts. Together, these findings establish the NTD as a critical regulator that enhances Ca2+ responsiveness by shifting Sorcins conformational equilibrium toward the active state and suggest that flexible N-terminal extensions regulate signaling within the penta-EF-hand protein family.

biophysics↗

bioRxiv2026

Benchmarking Deep Learning Predictions of Mutation-Induced Fold Switching

Many proteins are known to adopt multiple distinct folded states which are often associated with key functional behavior. A predictive understanding of the properties of such fold-switching or metamorphic proteins can provide insights into protein dynamics and energetics, and enable the design of complex protein functions and molecular machines. Recently developed deep learning modeling tools, including AlphaFold, have led to dramatic increases in accuracy for prediction of protein structures from sequence, but their performance for prediction of point mutant effects or fold switching is unclear. Here we present a systematic NMR-characterized dataset of mutants of the GA/GB model fold-switching system and use it to evaluate whether current structure prediction and design methods can predict mutation-induced changes in fold state. We measured fold-state populations for variants at three key positions that differentially stabilize the 3, 4{beta}+, mixed, or unfolded states, generating a quantitative experimental benchmark for mutation-level fold switching. Using this benchmark to assess and compare a panel of deep learning and physics-based modeling and design algorithms, we found that this benchmark revealed variable and position-dependent performance across methods, with certain AlphaFold2-based algorithms were able to predict mutant effects at individual sites, indicating some understanding of physical effects of residue substitutions. Additional comparisons of predictions with experimentally measured stability changes further highlighted position-dependent success and general challenges for predictive algorithms. Together, this study provides a new benchmark for mutation-induced fold switching and reveals the current capabilities and limitations of deep learning models for predicting mutation-dependent protein conformational states.

biophysics↗

bioRxiv2026

Engineered subtilisin protease degrades active KRAS in cancer cells, leading to differential cell targeting

Controlling aberrant RAS signaling has been the subject of intensive efforts aimed at developing specific RAS inhibitors, small molecules that promote RAS degradation, and monobodies that inhibit RAS activity. Direct proteolytic degradation of RAS by site-specific proteases has received considerably less attention. A naturally-occurring protease from Vibrio vulnificus toxin cleaves all RAS isoforms at switch I and attenuates RAS signaling in cell models and patient-derived xenografts, thus demonstrating the potential of this approach. We previously designed a RAS-specific protease, called RASProtease (or RASp), that site-specifically cleaves RAS at switch II. Attacking switch II leverages an order to disorder transition that this region undertakes upon conversion to the active form that predominates in cancer. Switch II participates in an allosteric network that controls KRAS oncogenicity, making it a promising target for proteolytic cleavage that modulates RAS signaling. Preferential targeting of active RAS could be particularly useful for studying RAS signaling networks as well as having potential therapeutic value. Here we examined the effects of RASp cleavage on downstream signaling and cell viability in the MIA PaCa-2 cancer cell model, which harbors homozygous KRAS G12C and is KRAS-dependent for growth and survival. We found that cleavage of KRAS G12C coincided with a decrease in MEK-ERK signaling and resulted in extensive MIA PaCa-2 cell death 24 hours after induction of RASp expression. This level of cell death far exceeded that of control HEK 293T cells under the same conditions, underscoring the vulnerability of this cancer cell model to KRAS G12C elimination.

cancer biology↗

bioRxiv2026

Substrate specificity in a designed RAS-targeting protease is coupled to active site and distal motions

Designing proteases with tailored substrate specificity has emerged as a powerful strategy for manipulating protein function in cells. RAS, a key regulator of cell survival and proliferation, is a compelling target for such approaches. Mutations in RAS are involved in about one-third of all human cancers and drive the hyperactive signaling that promotes tumorigenesis, growth, and metastasis in cancers such as pancreatic and lung cancer. This creates a pressing need for strategies capable of modulating mutant RAS with high substrate specificity to avoid unintended cleavage events. As a model for targeted proteolysis, we present the high-resolution crystal structures of RASProtease(II), which provide a detailed view of the enzymes active site and substrate-binding architecture. Kinetic experiments showed that cleavage of the cognate QEEYSAM substrate is approximately 30-fold faster than the non-cognate QEEISAM, demonstrating strong proteolytic selectivity. NMR dynamics studies combined with structural mapping revealed that substrate binding modulates not only the active site, but also distal regions of RASProtease(II), uncovering long-range allosteric networks. Contrary to the conventional view that non-cognate substrates are simply poor fits for the active site, we found that binding of the non-cognate peptide induces a greater amount of conformational dynamics in the protease than in the apo form or cognate complex, resulting in significant destabilization and providing a mechanistic explanation for the reduced catalytic efficiency. These results reveal how distal structural networks help define substrate specificity and provide principles for rationally designing proteases with enhanced specificity for therapeutic applications. Significance StatementDesigner proteases are gaining interest for their potential as therapeutic agents. It is therefore critical to understand the mechanisms that differentiate cognate and non-cognate sequences to minimize deleterious off-target cleavage events. While the conventional view is that non-cognate substrates are simply poor fits for the active site, we demonstrate here that dynamic allostery plays an integral role in distinguishing between cognate and off-target sequences, using a protease designed to target the oncoprotein RAS as our model system. Moreover, because the RASProtease is derived from a prototypical serine protease, subtilisin, it is likely that the overall mechanism employed for selecting cognate over non-cognate substrates presented here will be generalizable, providing principles for designing enhanced specificity in a wide range of proteases.

biochemistry↗

bioRxiv2025

Proteome-wide identification of metamorphic protein candidates using mass spectrometry

How protein folds remodel at low temperature remains poorly mapped at the proteome scale. We combine brief, temperature-controlled proteolysis with LC-MS/MS to chart cold-dependent conformational changes in Escherichia coli lysate. Methodological validation with the metamorphic protein Sa1V90T--whose /{beta}-plait and 3 states interchange between 30 {degrees}C and 5 {degrees}C--showed that a high-protease, short-digestion regime detects temperature-dependent exposure at four peptides, whereas the temperature-insensitive variant Sa1V90TV52D is largely unchanged. Applied to lysate, the workflow identifies >750 proteolytic sites across >250 abundant proteins (>10% of the identified proteome) whose local susceptibility shifts as temperature decreases from 35 {degrees}C to 5 {degrees}C, including five Sa1V90T peptides recapitulating purified-protein behavior. A site-level scoring model yields 338 destabilized peptides (177 proteins) and 426 stabilized peptides (138 proteins). Cold stabilization prominently marks translation machinery (>15 ribosomal proteins), while cooling also exposes regions in chaperones and cell-division factors. Sequence analysis links temperature sensitivity to charge-enriched composition, consistent with thermodynamic expectations. These data provide a proteome-wide view of cold-sensitive folding, implicate translation and cytokinetic systems in low-temperature remodeling, and illuminate a new strategy for identifying metamorphic proteins on an unprecedented scale.

biophysics↗

bioRxiv2025

Conformational dynamics and multi-modal interaction of Paxillin with the Focal Adhesion Targeting Domain

Paxillin (PXN) and focal adhesion kinase (FAK) are two major components of the focal adhesion complex, a multiprotein structure linking the intracellular cytoskeleton to the cell exterior. The interaction between the disordered N-terminal domain of PXN and the C-terminal targeting domain of FAK (FAT) is necessary and sufficient for localizing FAK to focal adhesions. Furthermore, PXN serves as a platform for recruiting other proteins that together control the dynamic changes needed for cell migration and survival. Here, we show that the PXN N-domain undergoes significant compaction upon FAT binding, forming a 48-kDa multi-modal complex with four major interconverting states. Although the complex is flexible, each state has unique sets of contacts involving disordered regions that are both highly represented in ensembles and conserved. PXN being a hub protein, the results provide a structural basis for understanding how shifts in the multi-state equilibrium (e.g. through ligand binding and phosphorylation) may rewire cellular networks leading to phenotypic changes. TeaserThe fuzzy complex between Paxillin and Focal adhesion targeting domain is resolved, with functional and translational implications.

biophysics↗

bioRxiv2021

Rules for designing protein fold switches and their implications for the folding code

We have engineered switches between the three most common small folds, 3, 4{beta}+, and /{beta}-plait, referred to here as A, B, and S, respectively. Mutations were introduced into the natural S protein until sequences were created that have a stable S-fold in their longer ([~]90 amino acid) form and have an alternative fold (either A or B) in their shorter (56 amino acid) form. Five sequence pairs were designed and key structures were determined using NMR spectroscopy. Each protein pair is 100% identical in the 56 amino acid region of overlap. Several rules for engineering switches emerged. First, designing one sequence with good native state interactions in two folds requires care but is feasible. Once this condition is met, fold populations are determined by the stability of the embedded A- or B-fold relative to the S-fold and the conformational propensities of the ends that are generated in the switch to the embedded fold. If the stabilities of the embedded fold and the longer fold are similar, conformation is highly sensitive to mutation so that even a single amino acid substitution can radically shift the population to the alternative fold. The results provide insight into why dimorphic sequences can be engineered and sometimes exist in nature, while most natural protein sequences populate single folds. Proteins may evolve toward unique folds because dimorphic sequences generate interactions that destabilize and can produce aberrant functions. Thus, two-state behavior may result from natures negative design rather than being an inherent property of the folding code. Significance StatementWe establish general rules for designing protein fold switches by engineering dimorphic sequences that link the three most common small folds. The fact that switches can be engineered in arbitrary and common protein folds, sheds light on several important questions: 1) What is the generality of fold switching? 2) What types of folds are amenable to switching? 3) What properties are shared by sequences that can fold into two completely different structures? This work has implications for understanding how amino acid sequence encodes structure, how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure. ClassificationBiological Sciences: Biochemistry; Physical Sciences: Biophysics and Computational Biology

biophysics↗

bioRxiv2020

Engineering protein-specific proteases: targeting active RAS

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

biochemistry↗

bioRxiv2019

A Non-genetic Mechanism for Chemoresistance in Lung Cancer: The Role of Integrin β4/Paxillin Axis

Tumor heterogeneity and cisplatin resistance are a major cause of tumor relapse and poor survival. Here we show that in lung adenocarcinoma (LUAD), paxillin (PXN) and integrin beta 4 (ITGB4) are associated with tumor progression, and cisplatin resistance. Silencing PXN and ITGB4 render cisplatin tolerant cells sensitive, and immunologically neutralizing ITGB4 improves sensitivity. The N-terminal half of PXN is intrinsically disordered and interacts with ITGB4 to regulate expression of USP1 and VDAC1 which are required for maintaining genomic stability and mitochondrial function in LUAD. By virtual screening an FDA-approved compound library, we identified compounds that interact with PXN in silico and attenuate cisplatin resistance in LUAD cells. RNAseq analysis identified a double negative feedback loop between ITGB4 and microRNA miR-1-3p, suggesting that bistability could lead to stochastic switching between cisplatin-sensitive and resistant states in these cells. The data highlight an alternate, non-genetic, mechanism underlying chemoresistance in lung cancer.

cancer biology↗

bioRxiv2018

PAGE4 and Conformational Switching: Insights from Molecular Dynamics Simulations and Implications for Prostate Cancer

Prostate-Associated Gene 4 (PAGE4) is a disordered protein implicated in the progression of prostate cancer. PAGE4 can be phosphorylated at two residue sites by Homeodomain-Interacting Protein Kinase 1 (HIPK1) to facilitate its binding to the Activator Protein-1 (AP-1) transcription factor. In contrast, a further hyperphosphorylation of PAGE4 by CDC-Like Kinase 2 (CLK2) reduces its binding affinity to AP-1, thus affecting the androgen receptor (AR) activity. Both SAXS and smFRET experiments have shown a structural expansion of PAGE4 upon hyperphosphorylation and a significant increase in size at its N-terminal half than that at its C-terminus. To understand the molecular mechanism underlying this structural transition, we performed a series of constant temperature molecular dynamics simulations using Atomistic AWSEM -- a multi-scale molecular model combining detailed atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interaction drives a transient formation of an N-terminal loop, which causes the change in size for different phosphorylated forms of PAGE4. Phosphorylation also changes the preference of secondary structure formation of PAGE4, which signifies a transition between states that display different degree of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions of different forms of PAGE4 with AP-1 and AR, a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4 and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching among different forms of PAGE4 may potentially affect the efficiency of therapeutic targeting of AR.

biophysics↗