Dorothee Kern

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 β-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.

Genetically encoded biosensors with changes in fluorescence lifetime (as opposed to fluorescence intensity) can quantify small molecules in complex contexts, even in vivo. However, lifetime-readout sensors are poorly understood at a molecular level, complicating their development. Although there are many sensors that have fluorescence-intensity changes, there are currently only a few with fluorescence-lifetime changes. Here, we optimized two biosensors for thiol-disulfide redox (RoTq-Off and RoTq-On) with opposite changes in fluorescence lifetime in response to oxidation. Using biophysical approaches, we showed that the high-lifetime states of these sensors lock the chromophore more firmly in place than their low-lifetime states do. Two-photon fluorescence lifetime imaging of RoTq-On fused to a glutaredoxin (Grx1) enabled robust, straightforward monitoring of cytosolic glutathione redox state in acute mouse brain slices. The motional mechanism described here is probably common and may inform the design of other lifetime-readout sensors; the Grx1-RoTq-On fusion sensor will be useful for studying glutathione redox in physiology.

Transition-state (TS) 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 TSs would pose a huge entropic bottleneck for enzyme catalysis. To shed light on this question, we studied the nature of the enzymatic TS 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 TSs, 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. TSEs as a key for efficient enzyme catalysis further boosts a unifying concept for protein folding and conformational transitions underlying protein function.

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 toward 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 p38α ΜΑP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we identified 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 promote p38α dephosphorylation. Our X-ray crystal structures of phosphorylated p38α 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 p38α 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 unique approach to achieving improved potency and specificity for therapeutic kinase inhibitors.

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
. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) states. 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
isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase Cyclophilin A. 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 found two additional properties of KaiB's landscape. First, the Ground state experiences cold denaturation: At 4 °C, the PD state becomes the majorly populated state. Second, 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 relieves buried charged residues, and support this structure experimentally. These results provide mechanistic insight into how evolution can design a single-sequence that achieves specific timing needed for its function.

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 finding 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 Evolutionary Scale Modeling (ESM-2). We demonstrate by use of a "categorical Jacobian" calculation that ESM-2 stores statistics of coevolving residues, analogously to simpler modeling 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.

AlphaFold2 (ref. 
) has revolutionized structural biology by accurately predicting single structures of proteins. However, a protein's biological function often depends on multiple conformational substates
, and disease-causing point mutations often cause population changes within these substates
. We demonstrate that clustering a multiple-sequence alignment by sequence similarity enables AlphaFold2 to sample alternative states of known metamorphic proteins with high confidence. Using this method, named AF-Cluster, we investigated the evolutionary distribution of predicted structures for the metamorphic protein KaiB
and found that predictions of both conformations were distributed in clusters across the KaiB family. We used nuclear magnetic resonance spectroscopy to confirm an AF-Cluster prediction: a cyanobacteria KaiB variant is stabilized in the opposite state compared with the more widely studied variant. To test AF-Cluster's sensitivity to point mutations, we designed and experimentally verified a set of three mutations predicted to flip KaiB from Rhodobacter sphaeroides from the ground to the fold-switched state. Finally, screening for alternative states in protein families without known fold switching identified a putative alternative state for the oxidoreductase Mpt53 in Mycobacterium tuberculosis. Further development of such bioinformatic methods in tandem with experiments will probably have a considerable impact on predicting protein energy landscapes, essential for illuminating biological function.

SignificanceWhile immensely successful, drugging kinases by active site inhibitors has faced major challenges. Selectivity issues leading to side effects and emergence of resistance mutations rendered treatments targeting active sites ineffective. Double-drugging via active and allosteric sites is a recently developed approach to overcome these obstacles. Using Aurora A and Abelson kinase, we provide a quantitative biophysical evaluation of double-drugging by rationally selecting inhibitor combinations with positive cooperativity. The results shed light on the interplay of kinase conformational equilibria and inhibitor-dose requirements for effective inhibition. Due to our rational selection of a positively cooperative drug combination for Abl, we deliver a fully closed, inactive Abl structure, including regulatory SH3 and SH2 domains. Collectively, this biophysical framework aids future rational double-drug designs.
Selective 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, Förster 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 cooperativity. 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 double-drugged kinase complexes reveal the molecular principles underlying the cooperative nature of double-drugging AurA and Abl with orthosteric and allosteric inhibitors. Finally, we observe a fully closed conformation of Abl when bound to a pair of positively cooperative orthosteric and allosteric modulators, shedding light on the puzzling abnormality of previously solved closed Abl structures. Collectively, our data provide mechanistic and structural insights into rational design and evaluation of double-drugging strategies.

Macromolecular function frequently requires that proteins change conformation into high-energy states. However, methods for solving the structures of these functionally essential, lowly populated states are lacking. Here we develop a method for high-resolution structure determination of minorly populated states by coupling NMR spectroscopy-derived pseudocontact shifts (PCSs) with Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (PCS-CPMG). Our approach additionally defines the corresponding kinetics and thermodynamics of high-energy excursions, thereby characterizing the entire free-energy landscape. Using a large set of simulated data for adenylate kinase (Adk), calmodulin and Src kinase, we find that high-energy PCSs accurately determine high-energy structures (with a root mean squared deviation of less than 3.5 angström). Applying our methodology to Adk during catalysis, we find that the high-energy excursion involves surprisingly small openings of the AMP and ATP lids. This previously unresolved high-energy structure solves a longstanding controversy about conformational interconversions that are rate-limiting for catalysis. Primed for either substrate binding or product release, the high-energy structure of Adk suggests a two-step mechanism combining conformational selection to this state, followed by an induced-fit step into a fully closed state for catalysis of the phosphoryl-transfer reaction. Unlike other methods for resolving high-energy states, such as cryo-electron microscopy and X-ray crystallography, our solution PCS-CPMG approach excels in cases involving domain rearrangements of smaller systems (less than 60 kDa) and populations as low as 0.5%, and enables the simultaneous determination of protein structure, kinetics and thermodynamics while proteins perform their function.