Biology subjects

Kern, D.

Publications and source records attributed to Kern, D..

18 recordsLinked to original sources

bioRxiv2026

Graphene Device for High-Temperature Crystallography Visualizes How Enzymatic Reactivity is Rooted in Ensemble Probability

Life exists at temperatures ranging from -20 to 122 {degrees}C. However, the majority of high-resolution structural data in the Protein Data Bank (PDB) were obtained at cryogenic temperatures, where biological function is halted due to the lack of thermal fluctuations. To overcome this fundamental problem and directly link structure to biological function, we have created a graphene-based device that significantly extends the temperature range for high-resolution macromolecular X-ray diffraction data collection. Using the new device, we obtained models of the transition state ensembles for a psychrophilic, a mesophilic, and a thermophilic homolog of the enzyme orotidine 5-monophosphate decarboxylase from -173 to 65 {degrees}C. The data reveal how the active site ensemble structure at the transition state of each homolog changes with temperature, directly visualizing how the measured catalytic rates are rooted in the ensemble probabilities of reactive distances. The multi-temperature transition state ensembles further illuminate why cryogenic data, although useful, are inaccurate for describing biological processes.

biophysics↗

bioRxiv2025

Comparing the evolvability of an ancestrally reconstructed and modern adenylate kinase

Directed evolution transformed protein engineering by providing a customizable framework for generating enzymes with improved catalytic performance across diverse functions. Yet modern enzymes often stall during directed evolution because populations become trapped on local fitness peaks. Researchers have suggested that ancestral enzymes offer better starting points because they are typically more thermostable. Here we propose and experimentally test an alternative explanation that does not depend on ancestral thermostability. We posit that ancestrally reconstructed sequences are unusually evolvable because they are inferred from the evolutionary lineages that survived to produce extant proteins. Less evolvable ancestors, and the trajectories emanating from them, disappeared by extinction and therefore do not contribute to reconstructed ancestors. Using thermophilic ancestral and modern adenylate kinases, we performed independent single-round selection experiments for activity in vivo and in vitro. In both settings, the ancestral enzyme tolerates a larger number of mutations, yielding more viable variants with greater genetic diversity than its modern descendants. Because mutational robustness promotes evolvability, these results support an intrinsic evolvability of reconstructed ancestral sequences that makes them superior launch points for directed evolution.

biochemistry↗

bioRxiv2025

Phosphatase SHP2 pathogenic mutations enhance activity by altering conformational sampling

SH2 domains are critical mediators of cellular signaling, although the molecular mechanisms by which they bind their phosphopeptide ligands remain incompletely understood. We investigate the atomic mechanisms underlying both healthy regulation and dysregulation of the human protein tyrosine phosphatase SHP2, a key regulator of cellular signaling. While most pathogenic mutations cluster near the PTP/N-SH2 interface, the E139D and T42A mutations are located within the regulatory SH2 domains, and their mechanisms of dysregulation remain controversial. The T42A mutation in the N-SH2 domain paradoxically increases phosphotyrosine-peptide binding affinity despite disrupting the hydrogen bond of T42 to the phosphoryl group, a puzzling contradiction that remains unresolved. We find that the T42A mutation shifts the conformational ensemble of peptide-bound N-SH2 toward a zipped {beta}-sheet state and suppresses millisecond conformational exchange, supporting a model in which enhanced stabilization of the zipped conformation contributes to hyperactivation. This conformational shift provides a structural rationale for the increased affinity of T42A and helps reconcile previously conflicting models of peptide-induced SHP2 activation. By integrating X-ray ensemble refinement with NMR relaxation, our work illustrates how complementary structural and dynamic approaches can uncover regulatory mechanisms in SHP2 and may inform broader principles of SH2-mediated phosphopeptide recognition. Significance StatementHere, we characterize how two pathogenic SH2-domain mutations alter SHP2 regulation and lead to hyperactivation. We identify a previously unobserved apo conformation of the N-SH2 domain in which Tyr66 occludes the peptide-binding cleft, indicating that a conformational change is required for full binding of activating phosphopeptides. Our data suggest that the T42A mutation shifts the equilibrium toward a zipped central {beta}-sheet state in the peptide-bound N-SH2 domain as the most likely model underlying the measured 10-fold increased binding affinity. These results help clarify the structural basis for SHP2 regulation and illustrate how conformational dynamics shape SH2-phosphopeptide recognition.

biophysics↗

bioRxiv2025

Learning millisecond protein dynamics from what is missing in NMR spectra

Many proteins biological functions rely on interconversions between multiple conformations occurring at micro-to millisecond ({micro}s-ms) timescales. A lack of standardized, large-scale experimental data has hindered obtaining a more predictive understanding of these motions. After curating >100 Nuclear Magnetic Resonance (NMR) relaxation datasets, we realized an observable for {micro}s-ms dynamics might be hiding in plain sight. Millisecond dynamics can cause NMR signals to broaden beyond detection, leaving some residues not assigned in the chemical shift datasets of ~10,000 proteins deposited in the Biological Magnetic Resonance Data Bank (BMRB)1. We made the bold assumption that residues missing assignments are exchange-broadened due to {micro}s-ms motions and trained various deep learning models to predict missing assignments. Strikingly, these models also predict exchange measured via NMR relaxation experiments, indicative of {micro}s-ms dynamics. The best of these models, which we named Dyna-1, leverages an intermediate layer of the multimodal language model ESM-32. Notably, dynamics directly linked to biological function, including enzyme catalysis and ligand binding, are particularly well predicted by Dyna-1, which parallels our findings that residues experiencing {micro}s-ms exchange are more conserved. We anticipate the datasets and models presented here will be transformative in unlocking the common language of dynamics and function.

biophysics↗

bioRxiv2025

The dramatic impact of the PER-DBT interaction on circadian timekeeping andtemperature compensation

Circadian timekeeping is orchestrated by multiple clock proteins, but mechanistic understanding is limited. AlphaFold predicts novel interactions between two helical domains of the Drosophila PERIOD (PER) protein and its major kinase Doubletime (DBT). These PER domains, SYQ and LT, are conserved with mammals and are predicted to interact identically with the DBT ortholog casein kinase 1 (CK1). SYQ and LT mutations designed to disrupt this interface result in severe period and temperature-sensitive fly phenotypes, and two LT mutations cause unprecedented free-running periods [≥] 44 hours and temperature-sensitive changes of [~]20 hours. Further characterization of a LT mutant strain shows a severe reduction in both PER phosphorylation and PER degradation. A human PER equivalent of the same LT mutation is a much less efficient in vitro substrate for CK1-mediated phosphorylation. These observations indicate that this conserved PER-DBT interaction is critical for PER phosphorylation, circadian timing and its enigmatic temperature compensation phenomena.

molecular biology↗

bioRxiv2024

A resource for comparing AF-Cluster and other AlphaFold2 sampling methods

Predicting multiple conformational states of proteins represents a significant open challenge in structural biology. Increasingly many methods have been reported for perturbing and sampling AlphaFold2 (AF2) [1] to achieve multiple conformational states. However, if multiple methods achieve similar results, that does not in itself invalidate any method, nor does it answer why these methods work. Interpreting why deep learning models give the results they do is a critically important endeavor for future model development and appropriate usage. To help the field continue to try to answer these questions, this work addresses misunderstandings and inaccurate conclusions in refs. [2-6]. Deep learning methods development moves quickly, and by no means did we think that the implementation of AF-Cluster in [7] would be the final word on how to sample multiple conformations. However, Porter et al.s primary critique, that AF-Cluster does not use local evolutionary couplings in its MSA clusters, is incorrect. We report here further analysis that underscores our original finding that local evolutionary couplings do indeed play an important role in AF-Cluster predictions, and refute all false claims made against [7].

biophysics↗

bioRxiv2024

The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale

How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We discovered two additional properties of KaiBs landscape. Firstly, the Ground state experiences cold denaturation: at 4{degrees}C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this "Enigma" state is a beta-strand register shift that eases buried charged residues, and support this structure experimentally. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function. Significance StatementOne can conceptualize KaiB as an on-off switch to regulate circadian rhythms in bacteria, where the "On state" is the Fold-switched state that binds KaiC and other proteins, and the "Off state" is the Ground state. Our work exemplifies how evolution tuned the kinetics of interconversion to align with the hour-long timescale of its biological function. The Ground state is dramatically destabilized at cold temperatures, and the system contains an alternate "off" conformation that exchanges with the primary "off" conformation at faster timescales than the rate-limiting step. Our findings demonstrate a simple principle for evolving a protein switch: one part of a protein domain remains stably folded to serve as a scaffold for the rest of the protein to re-fold.

biochemistry↗

bioRxiv2024

Dual-Action Kinase Inhibitors Control p38α MAP Kinase Threonine Dephosphorylation

Reversible protein phosphorylation directs essential cellular processes including cell division, cell growth, cell death, inflammation, and differentiation. Because protein phosphorylation drives diverse diseases, kinases and phosphatases have been targets for drug discovery, with some achieving remarkable clinical success. Most protein kinases are activated by phosphorylation of their activation loops, which shifts the conformational equilibrium of the kinase towards the active state. To turn off the kinase, protein phosphatases dephosphorylate these sites, but how the conformation of the dynamic activation loop contributes to dephosphorylation was not known. To answer this, we modulated the activation loop conformational equilibrium of human p38 MAP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we discovered three inhibitors that increase the rate of dephosphorylation of the activation loop phospho-threonine by the PPM serine/threonine phosphatase WIP1. Hence, these compounds are "dual-action" inhibitors that simultaneously block the active site and stimulate p38 dephosphorylation. Our X-ray crystal structures of phosphorylated p38 bound to the dual-action inhibitors reveal a shared flipped conformation of the activation loop with a fully accessible phospho-threonine. In contrast, our X-ray crystal structure of phosphorylated apo human p38 reveals a different activation loop conformation with an inaccessible phospho-threonine, thereby explaining the increased rate of dephosphorylation upon inhibitor binding. These findings reveal a conformational preference of phosphatases for their targets and suggest a new approach to achieving improved potency and specificity for therapeutic kinase inhibitors.

biochemistry↗

bioRxiv2024

Protein language models learn evolutionary statistics of interacting sequence motifs

Protein language models (pLMs) have emerged as potent tools for predicting and designing protein structure and function, and the degree to which these models fundamentally understand the inherent biophysics of protein structure stands as an open question. Motivated by a discovery that pLM-based structure predictors erroneously predict nonphysical structures for protein isoforms, we investigated the nature of sequence context needed for contact predictions in the pLM ESM-2. We demonstrate by use of a "categorical Jacobian" calculation that ESM-2 stores statistics of coevolving residues, analogously to simpler modelling approaches like Markov Random Fields and Multivariate Gaussian models. We further investigated how ESM-2 "stores" information needed to predict contacts by comparing sequence masking strategies, and found that providing local windows of sequence information allowed ESM-2 to best recover predicted contacts. This suggests that pLMs predict contacts by storing motifs of pairwise contacts. Our investigation highlights the limitations of current pLMs and underscores the importance of understanding the underlying mechanisms of these models. Significance StatementProtein language models (pLMs) have exhibited remarkable capabilities in protein structure prediction and design. However, the extent to which they comprehend the intrinsic biophysics of protein structures remains uncertain. We present a suite of analyses that dissect how the flagship pLM ESM-2 predicts structure. Motivated by a consistent error of protein isoforms predicted as structured fragments, we developed a completely unsupervised method to uniformly evaluate any protein language model that allows for us to compare coevolutionary statistics to older linear models. We further identified t hat E SM-2 a ppears to have a precise context size that is needed to predict inter-residue contacts. Our study highlights the current limitations of pLMs and contributes to a deeper understanding of their underlying mechanisms, paving the way for more reliable protein structure predictions.

bioinformatics↗

bioRxiv2023

Wide Transition-State Ensemble as Key Component for Enzyme Catalysis

Transition-state theory has provided the theoretical framework to explain the enormous rate accelerations of chemical reactions by enzymes. Given that proteins display large ensembles of conformations, unique transition states would pose a huge entropic bottleneck for enzyme catalysis. To shed light on this question, we studied the nature of the enzymatic transition state for the phosphoryl-transfer step in adenylate kinase by quantum-mechanics/molecular-mechanics calculations. We find a structurally wide set of energetically equivalent configurations that lie along the reaction coordinate and hence a broad transition-state ensemble (TSE). A conformationally delocalized ensemble, including asymmetric transition states, is rooted in the macroscopic nature of the enzyme. The computational results are buttressed by enzyme kinetics experiments that confirm the decrease of the entropy of activation predicted from such wide TSE. Transition-state ensembles as a key for efficient enzyme catalysis further boosts a unifying concept for protein folding and conformational transitions underlying protein function.

biophysics↗

bioRxiv2023

A biophysical framework for double-drugging kinases

Orthosteric inhibition of kinases has been challenging due to the conserved active site architecture of kinases and emergence of resistance mutants. Simultaneous inhibition of distant orthosteric and allosteric sites, which we refer to as "double-drugging", has recently been shown to be effective in overcoming drug resistance. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric modulators has not been undertaken. Here, we provide a quantitative framework for double-drugging of kinases employing isothermal titration calorimetry, Forster resonance energy transfer, coupled-enzyme assays, and X-ray crystallography. We discern positive and negative cooperativity for Aurora A kinase (AurA) and Abelson kinase (Abl) with different combinations of orthosteric and allosteric modulators. We find that a conformational equilibrium shift is the main principle governing this cooperative effect. Notably, for both kinases, we find a synergistic decrease of the required orthosteric and allosteric drug dosages when used in combination to inhibit kinase activities to clinically relevant inhibition levels. X-ray crystal structures of the doubledrugged kinase complexes reveal the molecular principles underlying the cooperative nature of double-drugging AurA and Abl with orthosteric and allosteric inhibitors. Finally, we observe the first fully-closed conformation of Abl when bound to a pair of positively cooperative orthosteric and allosteric modulators, shedding light onto the puzzling abnormality of previously solved closed Abl structures. Collectively, our data provide mechanistic and structural insights into rational design and evaluation of doubledrugging strategies.

biochemistry↗

bioRxiv2022

From primordial clocks to circadian oscillators

Circadian rhythms play an essential role in many biological processes and surprisingly only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and central component of the clock, with subsequent additions of KaiB and KaiA to regulate its phosphorylation state for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that possess just KaiBC, except for reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here, we investigate the primordial circadian clock in Rhodobacter sphaeroides (RS) that contains only KaiBC to elucidate its inner workings despite the missing KaiA. Using a combination X-ray crystallography and cryo-EM we find a novel dodecameric fold for KaiCRS where two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the C-terminal extension of KaiCRS and serves as an ancient regulatory moiety later superseded by KaiA. A coiled-coil register shift between daytime- and nighttime-conformations is connected to the phosphorylation sites through a long-range allosteric network that spans over 160 [A]. Our kinetic data identify the difference in ATP-to-ADP ratio between day and night as the environmental cue that drives the clock and further unravels mechanistic details that shed light on the evolution of self-sustained oscillators.

biochemistry↗

bioRxiv2022

Prediction of multiple conformational states by combining sequence clustering with AlphaFold2

AlphaFold2 (AF2) has revolutionized structural biology by accurately predicting single structures of proteins and protein-protein complexes. However, biological function is rooted in a proteins ability to sample different conformational substates, and disease-causing point mutations are often due to population changes of these substates. This has sparked immense interest in expanding AF2s capability to predict conformational substates. We demonstrate that clustering an input multiple sequence alignment (MSA) by sequence similarity enables AF2 to sample alternate states of known metamorphic proteins, including the circadian rhythm protein KaiB, the transcription factor RfaH, and the spindle checkpoint protein Mad2, and score these states with high confidence. Moreover, we use AF2 to identify a minimal set of two point mutations predicted to switch KaiB between its two states. Finally, we used our clustering method, AF-cluster, to screen for alternate states in protein families without known fold-switching, and identified a putative alternate state for the oxidoreductase DsbE. Similarly to KaiB, DsbE is predicted to switch between a thioredoxin-like fold and a novel fold. This prediction is the subject of future experimental testing. Further development of such bioinformatic methods in tandem with experiments will likely have profound impact on predicting protein energy landscapes, essential for shedding light into biological function.

biochemistry↗

bioRxiv2020

Bimodal evolution of Src and Abl kinase substrate specificity revealed using mammalian cell extract as substrate pool

The specificity of phosphorylation by protein kinases is essential to the integrity of biological signal transduction. While peptide sequence specificity for individual kinases has been examined previously, here we explore the evolutionary progression that has led to the modern substrate specificity of two non-receptor tyrosine kinases, Abl and Src. To efficiently determine the substrate specificity of modern and reconstructed ancestral kinases, we developed a method using mammalian cell lysate as the substrate pool, thereby representing the naturally occurring substrate proteins. We find that the oldest tyrosine kinase ancestor was a promiscuous enzyme that evolved through a more specific last common ancestor into a specific human Abl. In contrast, the parallel pathway to human Src involved a loss of substrate specificity, leading to general promiscuity. These results add a new facet to our understanding of the evolution of signaling pathways, with both subfunctionalization and neofunctionalization along the evolutionary trajectories.

biochemistry↗

bioRxiv2018

Dynamics of human protein kinases linked to drug selectivity

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.\n\neLife digestThe Ser/Thr kinase Aurora A is an important target for the development of new anticancer therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in a background of over 550 protein kinases with very similar structures. To this end, understanding the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational change after drug binding (i.e., induced-fit step) dictates drug affinity.

cancer biology↗

bioRxiv2017

Rescue of Conformational Dynamics in Enzyme Catalysis by Directed Evolution

Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes1-3. However, a comprehensive understanding of how evolution shapes the energy landscape of catalysis remains a fundamental challenge. Protein dynamics is widely recognized as important, but due to the inherent flexibility of biological macromolecules it is often difficult to distinguish which conformational changes are directly related to function. Here, we used directed evolution on an impaired mutant of the human proline isomerase cyclophilin A (CypA) and identify two second-shell mutations that partially restore its catalytic activity. We show both kinetically, using NMR spectroscopy, and structurally, by room-temperature X-ray crystallography, how local perturbations propagate through a large allosteric network to facilitate conformational dynamics. The increased catalysis selected for in the evolutionary screen could be rationalized entirely by accelerated interconversion between the two catalytically essential conformational sub-states, which are both captured in the high-resolution X-ray ensembles at room temperature. Our data provide a glimpse of the evolutionary trajectory of an enzymes energy landscape and shows how subtle changes can fine-tune its function.

biophysics↗

bioRxiv2017

Ectopic activation of the Spindle Assembly Checkpoint reveals its biochemical design and physiological operation

Switch-like activation of the Spindle Assembly Checkpoint (SAC) is critical for accurate chromosome segregation during cell division. To determine the mechanisms that implement it, we engineered an ectopic, kinetochore-independent SAC activator, the \"eSAC\". The eSAC stimulates the SAC signaling cascade by artificially dimerizing the Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the signaling scaffold in the kinetochore. Quantitative analyses and mathematical modeling of the eSAC reveal that the recruitment of multiple SAC proteins by the KNL1 phosphodomain stimulates synergistic signaling, which enables a small number of KNL1 molecules produce a disproportionately strong anaphase-inhibitory signal. However, when multiple KNL1 molecules signal concurrently, they compete for a limited cellular pool of SAC proteins. This frustrates synergistic signaling and modulates signal output. Together, these mechanisms institute automatic gain control - inverse, non-linear scaling between the signal output per kinetochore and the unattached kinetochore number, and thus enact the SAC switch.

cell biology↗

bioRxiv2017

Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex, the core component of the kinetochore-microtubule interface, to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.

cell biology↗