Poster-Abstracts

My collaborators and I investigated the opening mechanism of the receptor-binding domain (RBD) of the glycosylated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) trimeric spike protein, which opens up to bind to the angiotensin-converting enzyme 2 (ACE2) receptor on a host cell to trigger infection, using molecular dynamics (MD) simulations [1]. Specifically, we used the weighted ensemble (WE) method [2], an enhanced sampling method for MD simulations that runs many short trajectories in parallel to efficiently sample the pathways of interest. This was the largest WE method all-atom simulation to date, involving ∼500,000 atoms. We sampled continuous pathways from closed to open for the glycosylated protein and from these pathways, we were able to identify important glycans (a type of sugar molecule) and residues that facilitate the RBD opening, which is an essential step for SARS-CoV-2 to infect human cells (Fig. 1). Our work was able to offer insight into the fundamental mechanisms of SARS-CoV-2 viral entry and infection, which could be crucial for researchers developing the next SARS-CoV-2 vaccines and treatments. This work was also recognized with the Gordon Bell Special Prize for High Performance Computing-Based COVID-19 Research, awarded by the Association for Computing Machinery (ACM) [3].

[1] Terra E Sztain, Surl-Hee Ahn, Anthony T Bogetti, Lorenzo Casalino, Jory A Goldsmith, Ryan S McCool, Fiona L Kearns, J Andrew McCammon, Jason S McLellan, Lillian Chong, et al. A glycan gate controls opening of the sars-cov-2 spike protein. bioRxiv, 2021.

[2] Gary A Huber and Sangtae Kim. Weighted-ensemble brownian dynamics simulations for protein association reactions. Biophysical Journal, 70(1):97–110, 1996.

[3] Lorenzo Casalino, Abigail C Dommer, Zied Gaieb, Emilia P Barros, Terra Sztain, Surl-Hee Ahn, Anda Trifan, Alexander Brace, Heng Ma, Hyungro Lee, et al. Ai-driven multiscale simulations illuminate mechanisms of sars-cov-2 spike dynamics. BioRxiv, 2020.

Authors/affiliations:

Terra Sztain^1†, Surl-Hee Ahn^1†, Anthony T. Bogetti³, Lorenzo Casalino¹, Jory A. Goldsmith⁴, Evan Seitz⁵, Ryan S. McCool⁴, Fiona L. Kearns¹, Francisco Acosta-Reyes⁶, Suvrajit Maji⁶, Ghoncheh Mashayekhi⁷, J. Andrew McCammon^1,2, Abbas Ourmazd⁶, Joachim Frank^5,6, Jason S. McLellan⁴, Lillian T. Chong³, Rommie E. Amaro¹

1. Department of Chemistry and Biochemistry, UC San Diego, La Jolla, CA 92093

2. Department of Pharmacology, UC San Diego, La Jolla, CA 92093

3. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260

4. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712

5. Department of Biological Sciences, Columbia University, New York, NY, 10032, USA

6. Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY 10032, USA

7. Department of Physics, University of Wisconsin-Milwaukee, 3135 N. Maryland Ave, Milwaukee, WI 53211, USA

† These authors contributed equally to this work

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolytetrimethylamineN-oxide(TMAO) issued by marine animals to counter act the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and ureadenaturation has been the subject of several studies, but the effect of TMAO against thermal unfolding has not been widely studied. The consensus for pressure stabilization is that TMAO preferentially stabilizes the collapsed/folded state. To delineate the effect of TMAO on folded and unfolded ensembles at different temperatures, we study the well characterized, fast-folding protein B(PRB). We have carried out extensive 150+s long all-atom simulations of thermal unfolding of PRBat multiple concentrations of TMAO. The simulations captured folding and unfolding events and show an increased stability of PRB in presence of TMAO. We hypothesize that a noverall smoothing of the protein energy landscape leads to a change in the population of intermediate ensembles. Quantifying TMAO-water interactions revealed that TMAO form sashell near but not at the protein surface, disrupting the water network and increasing hydration of the protein. Intriguingly, we found that there are intermittent interactions between TMAO and the protein side chains. Although previous studies have proposed such interactions, the long-time scales we study here help to highlight the protein’s sensitivity to local environment, particularly hydration, and raise questions about how even transient interactions could couple to TMAO effects on the solvent and yield overall stabilization of the protein structure

Authors/affiliations:

Mayank Boob1, Shahar Sukenik2, Martin Gruebele1-4andTaras Pogorelov1,2,4,5

¹Center for Biophysics and Quantitative Biology,University of Illinois at Urbana-Champaign,Champaign, IL 61801, United States.

²Department of Chemistry,University of Illinois at Urbana-Champaign,Champaign,IL61801,UnitedStates.

³Department of Physics and Center for the Physics of LivingCells,University of Illinois at Urbana-Champaign, Champaign, IL 61801, United States.

⁴Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign,Champaign, IL 61801, United States.

⁵National Center for Supercomputing Applications,University of Illinois at Urbana–Champaign,Urbana,IL 61801

X-ray free-electron lasers (XFELs) produce x-ray pulses with very high brilliance and short pulseduration. These properties allow to investigate the structure of tiny crystals and single biological particles and to resolve the dynamics of biomolecules down to the femtosecond timescale. To deliver the samples rapidly into the XFEL beam, liquid jets are used. However, the high intensity of the X-ray pulse leads to a vaporization and explosion of the liquid jet. The expanding gas launches shock wave trains traveling along the jet that can affect biomolecular crystals before they have beenprobed. We modeled the exposure of a nanometer sized water jet to an XFEL pulse by heating the center of a water jet to very high temperatures. Molecular Dynamics simulations performed with GROMACS2018.5 show that the heating induces an explosion of the jet. Analysis of the density in the jet revealed shock waves that form close to the explosion center and travel along the jet. A trailing shock wave formed after the first shock wave, similar to the shock wave trains in experiments. Although using a purely classical model in the simulations, the resulting explosion and shock wave dynamics and shock wave attenuation in our simulation are comparable to experiments.

Authors/affiliations:

Leonie Chatzimagas, Jochen S. Hub

Saarland University, Department of Theoretical Physics, Saarbrücken, Germany

Amphotericin (AmB) is a potent and effective drug to combat life-threatening fungal infections. However, it is extremely toxic to humans and can lead to fatal complications including kidney damage or heart failure. It was experimentally shown that AmB’s anti-fungal activity originates from its binding to ergosterol in fungal cells. Since ergosterol is similar to human cholesterol, the toxicity purported to come from AmB’s non-specific binding to both sterols. To develop AmB derivatives that maintain potency while reducing toxicity, structural elucidation of sterol interactions is necessary. The goal of our study is to characterize unique binding modes between sterols and AmB and reveal anatomistic mechanism behind its fungicidal activity. To this end, we use all-atom molecular dynamics (MD) simulations to capture how an NMR-derived AmB lattice structure interacts with the membrane bilayer. We efficiently explore the structural landscape of AmB-Sterol complexes using extensive ~80 μs of replica exchange MD(REMD). Resulting AmB membrane-embedded structures are then subjected to equilibrium MD with the highly mobile-mimetic (HMMM) model that uses a novelsterol-compatible, in-silico solvent. We connect our results to solid-state NMR experiments by calculating theoretical chemical shifts. Our methodology produced an ensemble of atomistically resolved dynamic AmB-Sterol complexes that are within 8ppm of experimental chemical shifts. We further support our computational model by showing structures of AmB complexes to satisfy within 3.5 Å the majority of distance restraints derived from NMR-REDOR experiments. This study offers a novel and generalizable workflow that combines distinct enhanced sampling methods, and bridges derived structures with NMR observables. Most importantly, the impact of this work will guide future experiments that focus on the design of AmB derivatives to selectively kill fungal cells and not to harm human cells. Such derivatives have the potential to save many lives.

Authors/affiliations:

Kevin J. Cheng2, Ashley M. De Lio¹, Agnieszka Lewandowska¹, Corinne P. Soutar¹,Martin D. Burke¹ Chad M. Rienstra³, Taras V. Pogorelov¹,²

¹Department of Chemistry, University of Illinois at Urbana-Champaign, Champaign, IL,USA

²Center for Biophysics, University of Illinois at Urbana-Champaign, Champaign,IL, USA

³Department of Chemistry, University of Wisconsin-Madison, Madison, WI,USA

Tripeptide loop closure (TLC) [1] is a standard procedure to reconstruct protein backbone conforma- tions, by solving a zero dimensional polynomial system yielding up to 16 solutions. In this work [2], we first show that multiprecision is required in a TLC solver to guarantee the existence and the accuracy of solutions. We then compare solutions yielded by the TLC solver against tripeptides from the Protein Data Bank. We show that these solutions are geometrically diverse (up to 3Å RMSD with respect to the data), and sound in terms of potential energy. Finally, we compare Ramachandran distributions of data and reconstructions for the three amino acids. The distribution of reconstructions in the second angular space (φ2,ψ2) stands out, with a rather uniform distribution leaving a central void.

We anticipate that these insights, coupled to our robust implementation in the Structural Bioinfor- matics Library (https://sbl.inria.fr/doc/Tripeptide_loop_closure-user-manual.html), will boost the interest of TLC for structural modeling in general, and the generation of conformations of flexible loops in particular.

[1] Evangelos A Coutsias, Chaok Seok, Matthew P Jacobson, and Ken A Dill. A kinematic view of loop closure. Journal of computational chemistry, 25(4):510–528, 2004.

[2] T. O’Donnell, C.H. Robert, and F. Cazals. Tripeptide loop closure: a detailed study of reconstructions based on Ramachandran distributions. Submitted, 2021.

Lytic polysaccharide monooxygenases (LPMOs) have a key role in degrading recalcitrant biomass, such as cellulose and chitin. Several LPMOs are multidomain proteins that contain carbohydrate-binding modules (CBMs). These CBMs are known to promote LPMO efficiency, but structural and functional properties of some of these CBMs remain unknown and it is not clear why some LPMOs, like CjLPMO10A from Cellvibrio japonicus, have two CBMs (CjCBM5 and CjCBM73). Chitin-binding experiments and molecular dynamics simulations show functional differences for both CBMs in terms of chitin binding. These differences correlate with different architectures of their substrate-binding surfaces.

Reference:

NMR structures and functional roles of two related chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus. Eva Madland, Zarah Forsberg, Yong Wang, Kresten Lindorff-Larsen, Axel Niebisch, Jan Modregger, Vincent G. H. Eijsink, Finn L. Aachmann, Gaston Courtade. bioRxiv 2021.04.25.441307; doi: https://doi.org/10.1101/2021.04.25.441307

Authors/affiliations:
Eva Madland*1 , Zarah Forsberg*2 , Yong Wang3 , Kresten Lindorff-Larsen3 , Axel Niebisch4 , Jan Modregger4 , Vincent G. H. Eijsink2 , Finn L. Aachmann1, Gaston Courtade1 * These authors contributed equally to this work
1 Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, 7491Trondheim, Norway
2 Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway
3 Structural Biology and NMR Laboratory, Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Denmark
4 Eucodis Bioscience GmbH, Campus Vienna Biocenter 2, 1030 Wien, Austria

Intrinsically disordered proteins (IDPs) adopt a wide variety of conformations in solution, without a distinct equilibrium structure. Here, we investigate the dynamics of IDPs, using the antimicrobial saliva protein Histatin 5 as model.A suitable technique for this purposeis quasi-elastic neutron scattering (QENS), which through the incoherent scatteringprobes the self-diffusion of particles onbiologically relevantlength-andtimescales.Here, focus is on the center-of-mass diffusion, consideringdynamics with respect to temperature and self-crowding effects. The diffusion obtained is a convolution of translational and rotational diffusion, but implicit relations between these are known. Therefore, atomistic molecular dynamics simulations previously performed are analyzed to compare with the experimentally achieved results., providing further insight into the dynamical properties of IDPsand how these are affected by self-crowding and temperature.

Authors/affiliations:

Eric Fagerberg,Sam Lenton, Tilo Seydel, Marie Skepö

Deeply intertwined with protein folding studies, the discovery and characterization of the different types of elementary interactions governing the conformational stability of proteins (i.e. the difference in free energy between the folded conformation and the ensemble of unfolded ones) has also be actively pursued. A wealth of experimental studies has stimulated the development of theoretical frameworks to explain the folding reaction, and of a myriad of bioinformatics tools to predict protein structure from sequence or the impact of point mutations leading to single amino acid variations (SAVs) on human disease, or to rationally design new proteins for biotechnological purposes. However, we still lack a quantitative understanding of protein stability as the thermodynamic properties that govern protein forms and their interaction capabilities cannot be calculated from first principles. This hampers the integrated description of cellular function and the rational exercise of protein design. We recently shed some light onto the issue of calculating protein thermodynamics from first principles by setting a quantitative semi-in-silico approach, which was tested on two two-state model proteins [1]. We now have extended the application of this method to new two-state proteins, and to more complex systems like a protein that folds through a three-state mechanism and one that binds a cofactor (holo-protein). We also have tested the effect of solvating conditions like pH, and of SAVs on the protein thermal stability, which resulted in quite impressive accuracy when compared to experimental data. In this work we demonstrate that a simple atomistic model of the unfolded ensemble of a protein, in combination with an X-ray or NMR structure of its folded state, and with existing force fields and water models, may allow to accurately calculate thermodynamics quantities like ΔHunf, ΔCpunf and ΔGunf. It may be of help to better predict the phenotype associated with genetic variations, to break down the contribution of the different elementary interactions to protein stability, or to further fine tune force fields and water models.

[1] Juan José Galano-Frutos and Javier Sancho; Journal of Chemical Information and Modeling 2019, 59 (10), 4350-4360.

Authors/affiliations:
Juan José Galano-Frutos^{1 2*} , Francho Nerin-Sanz¹ , Javier Sancho^{1 2 3
1} Department of Biochemistry, Molecular and Cell Biology. Faculty of Science, University of Zaragoza, Zaragoza 50009, Spain.
² Biocomputation and Complex Systems Physics Institute (BIFI). Joint Units BIFI-IQFR (CSIC) and GBs-CSIC, University of Zaragoza, Zaragoza 50018, Spain.
³ Aragon Health Research Institute (IIS Aragón), Zaragoza 50009, Spain

Transient receptor potential vanilloid 4 (TRPV4) is a eukaryotic ion channel involved in many physiological processes and diseases. Compared to most other ion channels, TRPV4 exhibits a highly pronounced polymodality, including various physical stimuli such as mechanical force, osmotic stress, and temperature, and binding of proteins, lipids, nucleotides, ions, or small molecule compounds. A hot spot for protein and lipid binding is the cytosolic TRPV4 N-terminal domain (NTD) comprising an α-helical ankyrin repeat domain (ARD) and a putatively intrinsically disordered region (IDR). The recently determined TRPV4 cryo-EM structure sheds light on how structural changes in the ARD upon ligand binding may propagate to the ion-conducting pore and thus regulate channel activity. The IDR, however, is missing in the cryo-EM structure. It thus remains unclear how binding events and structural changes in this region may control ion gating. We have used a combination of SAXS, NMR spectroscopy, and hydrogen/deuterium-exchange mass spectrometry to investigate the molecular structure of the TRPV4 NTD. Our studies revealed that the IDR is entirely disordered, as predicted by bioinformatic analysis, and behaves like a random chain. In isolation, the folded ARD is a highly dynamic protein that becomes structurally and dynamically stabilized upon adding IDR residues, thus indicating a yet undescribed coupling between a disordered and an ordered domain. Such coupling may link ligand binding in the IDR to conformational changes in the ARD and consequently in the rest of the ion channel. Our findings are a first step towards understanding how ligand binding in the IDR can be sensed in the rest of the TRPV4 channel and subsequently modulate ion gating

Authors/affiliations:

Benedikt Goretzki¹*, Frederike Tebbe ² , Cy Jeffries³, Ute A. Hellmich ^1,2

¹ Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University, Max-von-Laue-Strasse 9, 60438 Frankfurt, Germany

²Faculty of Chemistry and Earth Sciences, Institute of Organic Chemistry andMacromolecular Chemistry, Friedrich-Schiller-University, Humboldtstrasse 10, 07743 Jena,Germany

³ European Molecular Biology Laboratory (EMBL), Hamburg Outstation c/o DeutschesElektronen Synchrotron (DESY), Notkestrasse 85, D-22607, Hamburg, Germany

Recent studies suggest that the N-terminal region of α-synuclein (αS) plays a critical role for both the normal function and pathological dysfunction involved in Parkinson’s disease.

In this work, we perform expanded ensemble simulations for a comparative analysis between structural features of human αS and its Y39A, Y39F and Y39L variants. We show that removing aromatic functionality at position 39 of monomeric αS lead to protein variants populating more compact conformations, conserving its disordered nature and secondary structure propensities.

Contrasting with the subtle changes induced by mutations on the protein structure, removing aromaticity at position 39 impacts strongly on the interaction of αS with the potent amyloid inhibitor phthalocyanine tetrasulfonate (PcTS). Parallel-tempering well-tempered metadynamics simulations shed light on the nature of the binding on position 39, and its selectivity with other aromatic moieties in fragments of monomeric αS.

Our findings further support the role of Tyr-39 in forming essential inter and intramolecular contacts that might have important repercussions for the function and dysfunction of αS, and the nature of ligand binding in position 39 for αS.

Authors/affiliations:
Oscar Palomino-Hernandez#^{1 3 7 8}, Fiamma A. Buratti#,², Pamela S. Sacco², Giulia Rossetti^1,4,5, Paolo Carloni*^1,3,6, Claudio O. Fernández*,^2,9

¹Institute for Neuroscience and Medicine (INM-9) and Institute for Advanced Simulations (IAS-5), Forschungszentrum Jülich, 52425 Jülich, Germany.

²Max Planck Laboratory for Structural Biology, Chemistry and Molecular Biophysics of Rosario (MPLbioR, UNR-MPIbpC) and Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR, UNR-CONICET), Universidad Nacional de Rosario, Rosario, Argentina.

³Faculty of Mathematics, Computer Science and Natural Sciences, RWTH Aachen, 52425 Aachen, Germany.

⁴Department of Oncology, Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation University Hospital Aachen, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany.

⁵Jülich Supercomputing Center (JSC), Forschungszentrum Jülich, 52425 Jülich, Germany

⁶Institute for Neuroscience and Medicine (INM-11) Forschungszentrum Jülich, 52425 Jülich, Germany.

⁷Computation-based Science and Technology Research Center, The Cyprus Institute, Nicosia 2121, Cyprus.

⁸Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.

⁹Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.

^#These authors contributed equally to the manuscript.

Nuclear receptors are crucial transcription factors that respond to a broad range of external signaling ligands by binding cognate DNA response elements and thereby initiate transcription. As such, these proteins form tightly regulated, multi-component allosteric systems. RXRαis one of the key players in this protein family as it is involved in regulation of many genes by heterodimerization with other nuclear receptors. We employ single-molecule fluorescence techniques to investigate the conformational dynamics of RXRα’s ligand binding domain (LBD)—the essential regulatory domain. Ligand binding in known to “activate” the receptor, that is to increase its affinity towards coactivator recruitment. The activation of RXRα’s LBD is believed to be mediated by a conformational change of helix 12. We characterize the conformational ensemble of this helix and find an unexpected auto-repressed conformation where helix 12 occupies the coactivator binding site. This conformation remains highly populated even after ligand binding. We further investigate this unexpected mechanism of allosteric regulation. In particular, we address the questions: On what time scales do the conformational changes of helix12 take place? And how are its dynamics affected by modulations in other parts of RXRαand in its binding partners? Our studies will pave the way to better understanding of the complex regulatory machinery embodied in nuclear receptor function.

Authors/affiliations:

Jakub Jungwirth*,¹, Demian Liebermann¹, Yoav Barak², Inbal Riven¹, Gilad Haran¹

¹Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, Israel

²Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

The conformational heterogeneity of a folded protein can affect both its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2, and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here we have examined whether changed native state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics in the backbone and amino acid side chains with molecular dynamics simulations to quantify the native state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: Are ductionin conformational dynamics (and entropy) of the native state of L49I variant correlates with its decreased stability, while increased dynamics of the I57VandL49I/I57V variants correlates with their increased stability. Our further analysis of native CI2 dynamics using relaxation dispersion NMR experiments revealed that mutations can affect CI2 dynamics also on the μs-ms timescale. Similar to the fast dynamics, the changes on this times cale correlate well with the difference instability of CI2 variants. These findings suggest that explicitly accounting for changes in native state dynamics might be needed to improve the predictions of the effect of mutations on protein stability.

Authors/affiliations:

Yulian Gavrilov¹,³,FelixKümmerer¹,SimoneOrioli¹,²,AndreasPrestel¹,KrestenLindorff-Larsen¹,Kaare Teilum¹

¹Structural Biology and NMR Laboratory and the Linderstrøm-Lang Centre for Protein Science,Department of Biology, University of Copenhagen, Denmark

²Structural Biophysics, Niels Bohr Institute, Universityof Copenhagenm, Denmark

³Current address:Division of Biophysical Chemistry, Center for Molecular Protein Science, Department of Chemistry, Lund University, Sweden

Fatty acid photodecarboxylase (FAP) is one of the three enzymes discovered so far, whose catalytic activity requires a continuous flux of light [1]. FAP is involved in the metabolism of lipids in microalgae and catalyzes the decarboxylation of free fatty acids to alkanes or alkenes in response to blue light. A reported low-resolution crystal structure (3.2 Å) shows the fatty acid in a hydrophobic tunnel and its carboxylate group facing the light-capturing flavin adenine dinucleotide (FAD) [1]. The proposed photocycle remained poorly understood.

We used serial femtosecond crystallography (SFX) at an X-ray free electron laser (XFEL) to study FAP, allowing us to solve a radiation damage-free resting state structure and to study structural dynamics on the picosecond to microsecond timescale by time-resolved SFX (TR-SFX) at room temperature [2]. In particular, a pump-probe scheme permitted visualization of structural changes following reaction initiation by an optical pump pulse and subsequent XFEL probe pulses that generated diffraction patterns.

The radiation damage-free SFX structure of FAP’s resting state at 2 Å resolution confirmed the observation made in a high-resolution cryo-MX structure that unusual bending of the isoalloxazine ring of the FAD is present in the oxidized starting state. A TR-SFX study made it then possible to follow light-induced decarboxylation in real time.

Our TR-SFX results on FAP microcrystals complemented results that were obtained by a larger consortium (see authors of [3]) using macromolecular X-ray crystallography, fast and ultrafast absorption spectroscopy, time-resolved infrared spectroscopy, Fourier-transform infrared spectroscopy and simulation methods. Together, they provide a detailed view of FAP’s catalytic reaction and its intermediate states [3].

[1] Sorigué, D., Légeret, B., Cuiné, S., Blangy, S., Moulin, S., Billon, E., Richaud. P., Brugière, S., Couté, Y., Nurizzo, D., Müller, P., Brettel, K., Pignol, D., Arnoux, P., Li-Beisson, Y., Peltier, G., Beisson, F. (2017). An algal photoenzyme converts fatty acids to hydrocarbons. Science 357:903-907.

[2] Colletier, J-P., Schirò, G. & Weik, M. (2018). Time-Resolved Serial Femtosecond Crystallography, Towards Molecular Movies of Biomolecules in Action in X-ray Free Electron Lasers: A Revolution in Structural Biology. Fromme P, Boutet S, Hunter M Eds. Springer International Publishing, 11:331-356

[3] Sorigué D., Hadjidemetriou K., Blangy S., Gotthard S., Bonvalet A., Coquelle N., Samire P., Aleksandrov A., Antonucci L., Benachir A., Boutet S., Byrdin M., Cammarata M., Carbajo S., Cuiné S., Doak R. B., Foucar L., Gorel A., Grünbein M., Hartmann E., Hienerwadel R., Hilpert M., Kloos M., Lane T. J., Légeret B., Legrand P., Li-Beisson Y., Moulin S. L. Y., Nurizzo D., Peltier G., Schirò G., Shoeman R. L., Sliwa M., Solinas X., Zhuang B., Barends T. R. M., Colletier J.-P., Joffre M., Royant A., Berthomieu C., Weik M., Domratcheva T., Brettel K., Vos M. H., Schlichting I., Arnoux P., Müller P., Beisson F. (2021). Mechanism and dynamics of fatty acid photodecarboxylase. Science 372:eabd5687

Authors/affiliations:

K. Hadjidemetriou¹*, P. Arnoux², T. Barends³, F. Beisson², S. Blagny², S. Boutet^4, M. Byrdin¹, M. Cammarata⁵, S. Carbajo⁴, J.-P. Colletier¹, N. Coquelle6, B. Doak³, L. Foucar³, A. Gorel³, G. Gotthard⁷, M. Grünbein³, E. Hartmann³, M. Hilpert³, M. Kloss³, T.J. Lane⁴, P. Legrand⁸, D. Nurizzo⁷, A. Royant²,⁷, G. Schirò¹, I. Schlichting³ R. Shoeman³, M. Sliwa ⁹ , D. Sorigué², M. Weik¹

¹Institut de Biologie Structurale, Grenoble, France..

²Biosciences and Biotechnologies Institute of Aix-Marseille, Saint-Paul-lez-Durance, France.

³Max-Planck-Institut for Medical Research, Heidelberg, Germany.

⁴Linac Coherent Light Source, Menlo Park, USA.

⁵Department of Physics, University of Rennes, France.

⁶Institut Laue Langevin, Grenoble, France.

⁷European Synchrotron Radiation Facility, Grenoble, France

⁸Synchrotron SOLEIL, Gif-sur-Yvette, France.

⁹Laboratoire de Spectrochimie Infrarouge et Raman, Lille, France.

Plant adaptation in variable soil nitrate conditions involves nitrate transport genes that express pallets of nitrate transporters harboured on the membrane of the root cells. Of the distinct high and low affinity nitrate transporters, NRT1.1 acts both as high and a low affinity transporter, besides being a nitrate sensor. The mechanisms underlying multifaceted roles of NRT1.1, however, remain to be fully explored. Using the recently discovered X-ray crystallographic structure of NRT1.1, we provide structure based explaining to the adaptive nitrate transport function of NRT1.1. We report that local structural asymmetries between the monomers of the dimeric NRT1.1 provide functional basis to have dual-affinity nitrate binding capacity. Nitrate-binding to high-affinity monomer triggers conformational change, facilitating phosphorylation at a remote site near the dimer interface, through allosteric communication. Phosphorylation sets the dimer apart and the protein adapts high-affinity transport mode. Nitrate binding in both high and low affinity monomers, however, retains the unmodified NRT1.1 state and the protein adapts the low-affinity transport mode. Together these results advance our molecular understanding of adaptation in fluctuating nutrient availability and are a way forward for improving plant nitrogen use efficiency.

Authors/affiliations:

Rather Mubasher Rashid

Indian Institute of Science, Bangalore

Proton translocation across membranes for energy conversion and storage is vital to all kingdoms of life. It relies on characteristic proton flowsand modifications ofhydrogen bonding patterns, termed here protonation dynamics, in conjunction withstructural changes anddisplacements of water molecules.Fast magic anglespinning(MAS)NMR offers adirectview onto theunderlyinghydrogen exchange processes in membrane proteins.Here we demonstrate that reversible proton translocation happens already in the dark-stateof bacteriorhodopsin(BR), involving the retinal Schiff base (RSB), water 402, D85,andR82. MAS NMR shows exchangeable protonsin contact with D85 and at the RSB, the respective chemical shifts are in agreement with a carboxylicacidprotonat D85or hydrogen bonding to ahydronium ion 402as part of an equilibrium of tautomers.A preformed proton path is detected between D85 and the proton shuttle R82, indicating the presence of an active site proton cage and a possible proton transfer via R82.The protons at theD96and D85carboxylgroupsshow exchange with water molecules, in line withab initiomolecular dynamics simulations. We conclude that retinal isomerization makes the observed process irreversible and delivers a proton towards the extracellular release site

Authors/affiliations:
DanielFriedrich^1,2,5,6 Florian N. Brünig ³, Andrew J. Nieuwkoop^1,7, Roland R. Netz³, Peter Hegemann4andHartmut Oschkinat^1,2

¹Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany.

²Freie Universität Berlin, Institut für Chemie und Biochemie, 14195 Berlin, Germany.

³Freie Universität Berlin, Fachbereich Physik, 14195 Berlin, Germany

⁴Humboldt-Universität zu Berlin, Institut für Biologie, Invalidenstr. 42, 10115 Berlin, Germany.

⁵Present address: Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, MA 02138, USA

⁶Present address: Department of Cancer Biology, Dana-Farber Cancer Institute, 360 Longwood Avenue, Boston, MA 02215, USA

⁷Present address: Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 123 Bevier Road, Piscataway, NJ 08854, USA.

Many biological processes are executed by molecular machines, which efficiently convert chemical energy into mechanical motion and vice versa. In order to maintain function in steady-state and to tune the activity according to the cellular environment, the conformational motions of a machine’s domains occur in a cooperative manner via allosteric signalling pathways, which are embedded in the protein’s architecture. A well-studied example for such a machine is the tetradecameric chaperonin GroEL, which consumes ATP to promote the folding of substrate proteins to their native form. Surprisingly, while much is known about GroEL’s structure and function with respect to its many substrates and co-factors, a detailed dynamic description of its internal allosteric conformational changes and their typical time scales is still missing.

We report here the first single-molecule FRET experiments on the conformational dynamics of GroEL. FRET efficiency histograms depict how a GroEL subunit samples its conformational space in the presence of various substrates, such as nucleotides and its co-chaperone GroES. Interestingly, the histograms indicate conformational transitions that are fast on the measurement time (milliseconds). Indeed, analysis of the single-molecule data using a photon-by-photon hidden Markov modelling algorithm, H2MM, yields conformational transition rates ranging from 100-5000 s-1, even though the catalytic cycle of GroEL takes place on the timescale of seconds. We aim to understand the significance of these fast conformational motions for GroEL’s function. For that, we are targeting specific steps in the cycle by characterizing variants, defective in certain functional aspects, such as ATP hydrolysis or cooperative interactions between subunits. Our experiments allow us to describe synchronized motions and reveal the timescales of allosteric signal propagation within GroEL, with general implications for the operation principles of molecular machines.

Authors/affiliations:

Demian Liebermann¹,Gilad Haran¹, Amnon Horovitz¹

¹Weizmann Institute of Science, Rehovot 7610001, Israel

Atomistic ensembles are important in many areas of biology but are difficult to determine if the ensembles are disordered. By combining small-angle scattering (SAS) and molecular dynamics (MD) we are now able to determine such atomistic ensembles. SAS mayinvestigate biomolecules in a near-native environment, providing scattering curves I(q), but it is not appropriate to explain the scattering curve with only one structural model, obtained with experimental methods like NMR and X-ray crystallography, because proteins are highly dynamic in solutions. This is especially true for is flexible proteins. In such cases computational methods are needed, such as MD simulations.In various biophysical experiments, for example small-angle neutron scattering (SANS), heavy water or deuterium oxide can be used as a solvent. In order to model such experiments with MD simulations, pair potentials for heavy water are required that reproduce the known physicochemical differences relative to light water.We present an atomistic ensemble of a disordered Protein-RNA complex from SAXS and MDand three effective pair potentials for heavy water (SPC/E-HW, TIP3P-HW, and TIP4P/2005-HW).

[1] J. Macošek, S. Bernd, J.-B. Linse, S. Winter, J. Foot, K. Perez, M. Rettel, M.T. Ivanović, P. Masiewicz, B. Murciano, M.M. Savitski, J.S. Hub, F. Gabel, J. Hennig, Structure and dynamics of the quaternary hunchback mRNA translation repression complex,bioRxiv (2020), doi:10.1101/2020.09.08.287060[2]J.-B. Linse and J. S. Hub, Three-and Four-Site Models for Heavy Water: SPC/E-HW, TIP3P-HW, and TIP4P/2005-HW, chemrxiv(2021), doi:10.26434/chemrxiv.14229755.v1

Authors/affiliations:

Johanna-B. Linse¹, Jakub Macošek², Bernd Simon², Frank Gabel³, Janosch Hennig² and Jochen S. Hub¹

¹Theoretical Physics and Center for Biophysics, Saarland University, Saarbrücken 66123, Germany

²Structural and Computational Biology Unit, European Molecular Biology Laboratory Heidelberg, Heidelberg, 69117, Germany

³Institut Biologie Structurale, University Grenoble Alpes, CEA, CNRS, Grenoble, 38044, France

Protein-protein interaction (PPI) is one of the key regulatory features to drive biomolecular processes and hence is targeted for designing therapeutics against diseases. Small peptides are a new and emerging class of therapeutics owing to their high specificity and low toxicity. For achieving efficient targeting of the PPI, amino acid side chains are often stapled together resulting in the rigidification of these peptides. Exploring the scope of these peptides demands a comprehensive understanding of their working principle. In this work, two stapled p53 peptides have been considered to delineate their binding mechanism with mdm2 using computational approaches. The addition of stapling protects the secondary structure of the peptides even in the case of thermal and chemical denaturation. Although the introduction of a stapling agent increases the hydrophobicity of the peptide, the enthalpic stabilization decreases. This is overcome by the lowering of the entropic penalty and the overall binding affinity improves. The mechanistic insights into the benefit of peptide stapling can be adopted for further improvement of peptide therapeutics.

Authors
Atanu Maityϯ, Asha Rani Chowdhuryϯ, Rajarshi Chakrabarti

Muscles are materials structurally organised at multiple scales which our projet plan to take each into account inside a multiscale model. At the molecular level, muscles are mainly composed of two proteins, namely Myosin and Actin which interact to form a molecular motor acting in a cycle at the origin of muscular contraction. The energy used by this motor comes from the hydrolysis of ATP which leaves an ADP and an inorganic phosphate (Pi). We need to evaluate the free energy barriers associated with transitions between the steps of the cycle in order to later introduce these in a collective model of molecular motors. We present here the use of Molecular Dynamics to study one of the steps that involves the departure of the Pi from the active site from the Myosin VI cavity. We have perfomed Molecular Dynamics and particularly Umbrella Sampling simulations with the AMBER software to get the free energy barrier associated to the departure of Pi. We then have studied the trajectory from the simulation to analyse the path taken by Pi during departure.

Authors/affiliations:

Robin Manevy¹*, Matthieu Caruel²,³, Fabrice Detrez¹, Isabelle Navizet¹

¹MSME, Univ Gustave Eiffel, CNRS UMR 8208, Univ Paris Est Creteil, F-77454 Marne-la-Vallée, France

²Univ Paris Est Creteil, CNRS, MSME, F-94010 Creteil, France3 Univ Gustave Eiffel, MSME, F-77454 Marne-la-Vallée, France

³Univ Gustave Eiffel, MSME, F-77454 Marne-la-Vallée, France

Methyl groups are abundant high-resolution probes and reporters of protein structures and dynamics, which have been explored extensively by various spectroscopic techniques such as NMR and neutron scattering. Here, a new methyl-based toolkit for exploring protein structures is presented, which combines signal-enhancement by DNP (dynamic nuclear polarziation) with heteronuclear Overhauser effect (hetNOE), carbon–carbon-spin diffusion(SD) and strategically designed isotope-labeling schemes. It is demonstrated that within this framework, methyl groups can serve as dynamic sensors for probing local molecular packing within proteins. Furthermore, they can be used as “NMR torches” to selectively enlighten their molecular environment, e.g., to selectively enhance the polarization of nuclei within residues of ligand-binding pockets. Finally, the use of13C–13C spin diffusion enables probing carbon–carbon distances within the sub nanometer range, which bridges the so-called sub-nanometer distance gap between conventional13C-ssNMR methods and EPR spectroscopy. The applicability of these methods is directly shown on a large membrane protein, the light-driven proton pump green proteorhodopsin (GPR), which offers new insight into the functional mechanism of the early step of its photocycle

Authors/affiliations:

Jiafei Mao¹*, Björn Corzilius², Xiao He³, Clemens Glaubitz¹

¹ Institute of Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany

²Institut für Chemie, Dr.-Lorenz-Weg 2 and Forschungsgebäude "Science and Technology of Life,Light and Matter", Büro 208, Albert-Einstein-Straße 25, Universität Rostock, 18059 Rostock, Germany

³Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,School of Chemistry and Molecular Engineering, East China Normal University, and NYU-ECNU Centerfor Computational Chemistry at NYU Shanghai, Shanghai, 200062, P. R. China, Shanghai, 200062, P. R.China

Much of cell signaling occurs near the cell membrane, involving reactions between tethered enzymes. So far, most in vitro and in silico studies have considered tethered reactions without the significance of enzyme crowding, even though in vivo estimates expect crowding effects to be significant. In particular, a recent surface plasmon resonance experiment found that the binding rate of the enzyme SHP-1 to the tethered PD-1 decreases as the density of the system increases. We hypothesize this result is due to crowding effects. To quantify the significance of crowding effects on signaling biochemistry, we develop a computational method that efficiently simulates tethered reactions with crowding, by iterating between a Gillespie-style algorithm for reactions and a Metropolis-style algorithm for computing entropic and steric effects. We find that as enzyme size increases, dephosphorylation reactions increase, and they increase at a quicker rate. However, sizes greater than some critical size (which depends on the tether length) results in a decrease of dephosphorylation, and a slower reaction rate. This work provides unconsidered insight for studying tethered signaling processes

Authors/affiliations:

Trini Nguyen
University of California, Irvine

Studying lipid-protein interactions is fundamental to understand the complex behavior of biological membranes. In these systems, membrane proteins affect the organization of lipid bilayers and thus their mechanical properties, and vice-versa the local lipid environment alters membrane-protein function. Growing evidence supports the idea that lipid-protein interactions may modify not only the structure but also the dynamics of integral membrane proteins. The most abundant membrane protein in the eye lens is aquaporin-0 (AQP0), with reported roles in water conduction, cell-cell adhesion, and cell organization. Electron-crystallography (EC)and molecular dynamics (MD) simulations have contributed to our understanding of lipid-protein interactions by providing high-resolution three-dimensional structural and dynamical data for diverse lipidic environments in a systematic manner. We performed MD simulations to monitor the positioning of cholesterol around AQP0 in a sphingolipid membrane, closer to the natural environment of this protein, and examined the effect cholesterol concentration has on the localization of this sterol molecule. Additional pulling simulations indicate an effect of cholesterol on stability and organization for groups of tetramers. We thereby expand our previous studies focused on phospholipids by also considering cholesterol, which potentially plays a key role in the higher-order organization of AQP0 tetramers in the lens membrane.

Authors/affiliations:

Juan David Orjuela¹*, Po-Lin Chiu², Thomas Walz³, Bert L. de Groot⁴, Camilo Aponte-Santamaría¹,⁵

¹Max Planck tandem group in Computational Biophysics, University of Los Andes, 111711 Bogotá,Colombia

²School of Molecular Sciences, Arizona State University, Tempe, AZ 85287.

³Laboratory of Molecular Electron Microscopy, Rockefeller University, New York, NY, 10065

⁴Computational Biomolecular Dynamics group, Max Planck Institute for Biophysical Chemistry, D-37007 Göttingen, Germany.

⁵Molecular Biomechanics group, Heidelberg Institute for Theoretical Studies, 69118 Heidelberg,Germany.

Transient receptor potential vanilloid 4 (TRPV4) is a eukaryotic ion channel involved in manyphysiological processes and diseases. Compared to most other ion channels, TRPV4 exhibitsa highly pronounced polymodality, including various physical stimuli such as mechanical force,osmotic stress, and temperature, and binding of proteins, lipids, nucleotides, ions, or smallmolecule compounds. A hot spot for protein and lipid binding is the cytosolic TRPV4 N-terminaldomain (NTD) comprising an α-helical ankyrin repeat domain (ARD) and a putativelyintrinsically disordered region (IDR). The recently determined TRPV4 cryo-EM structure shedslight on how structural changes in the ARD upon ligand binding may propagate to the ion-conducting pore and thus regulate channel activity. The IDR, however, is missing in the cryo-EM structure. It thus remains unclear how binding events and structural changes in this regionmay control ion gating. We have used a combination of SAXS, NMR spectroscopy, andhydrogen/deuterium-exchange mass spectrometry to investigate the molecular structure ofthe TRPV4 NTD. Our studies revealed that the IDR is entirely disordered, as predicted bybioinformatic analysis, and behaves like a random chain. In isolation, the folded ARD is ahighly dynamic protein that becomes structurally and dynamically stabilized upon adding IDRresidues, thus indicating a yet undescribed coupling between a disordered and an ordereddomain. Such coupling may link ligand binding in the IDR to conformational changes in theARD and consequently in the rest of the ion channel. Our findings are a first step towardsunderstanding how ligand binding in the IDR can be sensed in the rest of the TRPV4 channeland subsequently modulate ion gating

Authors/affiliations:

Kevin Pounot^1*, Kevin Pounot^1,2, Giorgio Schiro¹, Martine Moulin³, Carlotta Marasini⁴, Daria Noferini⁵, Michaela Zamponi⁵, Michael Haertlein³, Trevor Forsyth³, Martin Blackledge¹, Bente Vestergaard⁴, Tilo Seydel², Annette E. Langkilde⁴, Douglas J. Tobias⁶, Martin Weik¹

¹Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38000 Grenoble, France

²Institut Max von Laue – Paul Langevin (ILL), CS 20156, F-38042 Grenoble, France

³Life Sciences Group, Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble, France

⁴Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark

⁵Jülich Centre for Neutron Science, outstation at Heinz Maier-Leibnitz Zentrum, Forschungszentrum Jülich GmbH,85747 Garching, Germany

⁶Department of Chemistry, University of California, Irvine, Irvine, California 92697, United States

Owing to the uncertainties of transport kinetics, the mechanism of net and exchange glucose transport via GLUT1 remains unclear, however extended atomistic molecular dynamics simulations of GLUT1 embedded in a fluid lipid membrane bilayer and surrounded by a physiological salt solution has resolved some of these ambiguities.

The first outstanding question is, does net glucose transport require conformational changes that alternately expose the central high affinity ligand binding site to externally and internally facing solutions, or does glucose transit by a random series of jumps between adjacent sites, aided by small fluctuations that randomly open and close the tunnels and cavities along the length of the central cleft?

By using a “flooding protocol” equivalent to 50 mM glucose in solution, molecular dynamics reveals a large number of amino-acids whose fluctuations alter with raised glucose concentration. These changes are most pronounced in the extra-membranous residues. With high solution glucose concentrations, the amplified fluctuations in GLUT1 allow glucose to permeate into the intramembranous regions. Glucose proximity to GLUT1 causes asynchronous expansions of bottlenecks occluding the internal and external openings of the central pore. This is accomplished by rotamer changes of large side chains of tyrosine, phenylalanine, and tryptophan residues, thereby permitting glucose and water to gain access to the central regions of the pore. Further, when glucose is close (< 6 Ǻ) to five salt bridges formed between polar residues, e.g. lysine or arginine and glutamate or aspartate, located at the external and internal openings of the central pore, the distance between these residues tends to increase.

With this flooding protocol, several glucose traversals through the central region, considered as the high affinity docking site have been observed. Except for the flooding protocol, the transporter does not display any sign of spontaneous glucose penetration into the intramembranous regions.

Another unresolved question relating to glucose exchange was investigated with a protocol, the “saturated” condition, where fifteen β-D- glucose ligands were initially docked along the length of the central pore, and their subsequent trajectories observed. Several mutual exchanges between adjacent ligands occurred, both at external and internal solution interfaces and within the internal vestibule. In the vestibule, these interchanges occur along the Z-axis, relatively slowly, ranging from 2- 20ns. These glucose exchanges demonstrate that accelerated exchange within GLUTs is consistent with a model for glucose transport, where glucose ligands can move stochastically within the various transporter tunnels and channels without aid of largescale conformational changes.

Authors/affiliations:

Saul Gonzalez-Resines¹, Peter J Quinn², Richard J Naftalin³, Carmen Domene^1,4

¹Departments of Chemistry University of Bath, Bath BA2 7AX, United Kingdom

²Department of Biochemistry, King’s College London, London, United Kingdom

³BHF Centre of Research Excellence, School of Medicine and Life Sciences, King’s College London

⁴Chemistry Research Laboratory, Mansfield Road, University of Oxford, Oxford OX1 3TA, United Kingdom

Viral infections constitute a major global health threat with the current coronavirus disease (COVID-19) outbreak caused by SARS-CoV-2 being the latest global pandemic. As part of our medicinal chemistry efforts to develop potential drugs with promise for clinical use,[1]a computational approach involving molecular docking studies on azole (triazole and pyrazole) based molecules on the virus main protease Mproand RNA polymerase[2]was conducted(Figure 1),as possible inhibitors that could be elected for further experimental bioassays. The protease and polymerase binding affinities and active site conformations of these azole derivatives 1-6were determined using autodock program. From molecular docking studies, these non-peptide new azole compounds exhibit possible Mpro inhibition activities with binding affinities (-4.7 kcal/mol to -6.5 kcal/mol) comparable to the recently reported peptide-like inhibitors such as α-ketoamide inhibitor 13b(-5.0 k/cal/mol). In addition, their binding affinities to RNA polymerase(-6.3 to -7.1 kcal/mol) were comparable to that of remdesivir (-6.6 kcal/mol).Notably, NMR spectroscopy studies on the bovine serum albumin(BSA)dynamics as a model protein revealed interactions between triazole 2and BSA, an important drug carrier protein.[3]A further analysis of the effect of the top score triazole 2with main protease using MD simulations showed good stability of the triazole-protease complex. Based on the observed binding energies and MD simulations/g_mmpbs are sults, these compounds may possess anti-viral bioactivity through inhibition of the virus main protease as well as RNA polymerase activities in living cells, and therefore need for further experimental assays. Keywords: Triazoles, Pyrazole, SARS-CoV-2, Main protease, Docking, RNA polymerase, MD Simulations.

REFERENCES

[1]aC. K. Rono, J. Darkwa, D. Meyer, B. C. Makhubela, Current Organic Synthesis 2019, 16, 900-912; bC. K. Rono, B. C. Makhubela, 2020.[2]R. Cannalire, C. Cerchia, A. R. Beccari, F. S. Di Leva, V. Summa, Journal of Medicinal Chemistry 2020.[3]J. Zhang, X. Gao, J. Huang, H. Wang, ACS omega 2020, 5, 16833-16840.

Authors/affiliations:

Charles K. Rono¹*, Banothile C.E. Makhubela,¹

¹Research Center for Synthesis And Catalysis, Department of Chemical Sciences, University of Johannesburg, 2006, South Africa.

Hydration-induced line narrowing in solid-state NMR spectra is a known phenomenon for softbiopolymer materials, such as elastin, collagen, silk. Here, we probe the increase of conformationaldynamics in protein-polymer films made from bioinspired tandem repeat proteins as they are ex-posed to water. Enhanced motion on the nanosecond timescale is probed by13C longitudinal relax-ation, while changes in microsecond timescale dynamics is assessed using13C rotating-frame relax-ation dispersion techniques. In particular, we introduce13C off-resonance Near Rotary-ResonanceRelaxation Dispersion (NERRD) method to identify the origin and quantify the amplitude ofthe fast microsecond motion. With the application of fast magic-angle spinning (55.55 kHz) andincreasing spin-lock field strengths approaching the half- and full-rotary-resonance conditions wedelineate the relaxation dispersion regime that reports on incoherent dynamics as opposed to co-herent dephasing of the transverse magnetization due to the presence of dense proton network.These relaxation measurements revealed that the motion of proline side-chain carbons are themost affected by hydration giving rise to sharp signals, and hence to long coherence life times, inboth dipolar-coupling-based and scalar-coupling-based 2D1H–13C experiments.

Authors/affiliations:

Romeo C. A. Dubini, Huihun Jung, Melik C. Demirel, and Petra Rov ́o

Faculty of Chemistry and Pharmacy, Department of Chemistry,Ludwig-Maximilians-Universitäat München

Enzymes are designed to accelerate vital chemical reactions by multiple orders of magnitude. Many of them harness large-scale motions of domains and subunits to promote their activity. Studying structural dynamics is hence essential to decipher how protein machines function. Combined with H²MM, a photon-by-photon hidden Markov model analysis,¹ single-molecule FRET studies on the enzyme adenylate kinase (AK) recently revealed very fast domain motions, two orders of magnitude faster than the turnover of the enzyme.² AK may use numerous cycles of conformational rearrangement in order to find a relative orientation of its substrates that is optimal for their chemical reaction.

In the present work, we studied how the ratio between the open and closed state of AK, particularly of the ATP-binding LID-domain, mediates the activity of the enzyme. High concentrations of the substrate AMP decrease the rate of LID-domain opening and disturb the balance between the two conformations. This effect is reflected in inhibition of enzymatic activity by high AMP levels. We manipulated the strength of substrate inhibition using several single-site mutations and could correlate the degree of substrate inhibition to changes in the open/closed ratio.

Interestingly, we also found that minor amounts of urea can partially revert the inhibitory impact of high AMP concentrations. Although urea commonly has been used as a protein denaturant, here, it actually enhances the catalytic activity of AK. Urea favors the open conformation of AK and can accordingly help to reestablish the optimal balance between domain opening and closing. However, in the absence of inhibitory concentrations of AMP, further promotion of the open state by urea is detrimental for turnover. This suggests a delicate balance between domain closure dynamics and substrate binding, which might not be a unique feature of AK. It is likely employed by a multitude of enzymes to regulate their activity.

1. Pirchi, M.; Tsukanov, R.; Khamis, R.; Tomov, T. E.; Berger, Y.; Khara, D. C.; Volkov, H.; Haran, G.; Nir, E., Photon-by-photon hidden Markov model analysis for microsecond single-molecule FRET kinetics. J. Phys. Chem. B 2016, 120 (51), 13065-13075.

2. Aviram, H. Y.; Pirchi, M.; Mazal, H.; Barak, Y.; Riven, I.; Haran, G., Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions. PNAS 2018, 115 (13), 3243-3248.

Authors/affiliations:

David Scheerer¹ *, Haim Yuval Aviram¹, Hisham Mazal^1,2, Inbal Riven¹ , Dorit Levy¹ , Gilad Haran¹

¹ Weizmann Institute of Science, 7610001 Rehovot, Israel

² Max-Planck Institute for the Science of Light, 91058 Erlangen, Germany.

Specific protein interactions typically require well-shaped binding interfaces. Here, we report a cunning exception. The disordered tail of the cell-adhesion protein E-cadherin dynamically samples a large surface area of the proto-oncogene β-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without dissociation of the complex. A few persistent interactions provide specificity whereas unspecific contacts boost affinity. The energy landscape of this complex is rugged with many small barriers (3 – 4 kBT) and reconciles specificity, high affinity, and extreme disorder. Given the roles of β-catenin in cell-adhesion, signaling, and cancer, this Velcro-like design has the potential to tune the stability of the complex without requiring dissociation.

Diffusion of an IDP on its folded ligand. Single-molecule FRET experiments and molecular simulations identify extreme disorder in the complex between E-cadherin (E-cad) and -catenin (-cat). The complex allows access to regulatory enzymes without requiring dissociation

References

[1] Diffusion of the disordered E-cadherin tail on β-catenin. Wiggers F., Wohl S., Dubovetsky A., Rosenblum G., Zheng W. and Hofmann H. (2021) bioRxiv 2021.02.03.429507

Authors/affiliations:

Felix Wiggers¹, Samuel Wohl², Artem Dubovetskyi¹, Gabriel Rosenblum¹,Wenwei Zheng³ and Hagen Hofmann^1
1Department of Structural Biology, Weizmann Institute of Science, Herzl St. 234, 76100 Rehovot, Israel

²Department of Physics, Arizona State University, Tempe, AZ 85287, USA, College of Integrative Sciences and Arts, Arizona State University, Mesa, AZ 85212, USA

The ultimate goal of structural biology is to link structure to function but the leap remains difficult based on a single in animate structure. Accordingly, the structural characterization of intermediate states is of high interest and pursued by many structural biology groups. With the advent of serial crystal lography at XFEL sand synchrotrons, time-resolved crystallography, performed following a specific perturbation of the crystalline system,should become feasible on numerous systems opening avenues to produce movies of proteins at work, thereby facilitating our understanding of their respective functions. However, alimitation remains in that the occupancy of the intermediate state has to be large enough to become visible in the electrondensity map. This is generally not the case ,with “perturbed” crystals existing as mixtures of initial, intermediate and final state(s). Differences between the “unperturbed ”and“ perturbed” dataset can be visualized in Fourier difference maps (Fobs,perturbed Fobs,unperturbed), which depict differences between the states. An even more powerful approach is to generate extrapolated structure factor amplitudes(Fextr,perturbed)1 solely describing the intermediate state. Such data processing has in the past been performed by some well-experienced crystallographers but remains out of reach for a wide audience. Here, we will present a user-friendly program,written in python and exploiting the cctbx toolbox modules, that allows the calculation of high-quality Fourier difference maps, estimation of the occupancy of the intermediate state(s) in the crystals, and generation of extrapolated structure factor amplitudes. Briefly, the program uses Bayesian statistics to weight structure factor amplitude differences 2 which are then used to generate extrapolated structure factor amplitudes for a range of possible intermediate state occupancies, with distinct weighting schemes. 1,3 Based on the comparis on between experimental and calculated differences, the correct occupancy of the intermediate state is determined and its structure refined, shedding light on conformational changes not visible before. With various user-controllable parameters of which defaults are carefully chosen, the program is adapted to be used by a wide audience of structural biologists, ranging from well-experienced crystallographers to newcomers in the field. We anticipate that this program will ease and accelerate the handling of time-resolved structural data, and thereby the understanding of molecular processes underlying function in a variety of proteins.

[1]Genick,U..,Borgstahl,G.E.,Ng,K.,Ren,Z.,Pradervand,C.,Burke,P.M.,Srajer,V.,Teng,T.Y.,Schildkamp,W.,McRee,D.E.,Moffat,K.&Getzoff,E.D.(1997),Science,275,1471-1475.[2]Ursby,T.&Bourgeois,D.(1997),ActaCrystallogr.Sect.A,53,564-575.[3]Coquelle,N.,Sliwa,M.,Woodhouse,J.,andothers,Colletier,J.-P.,Schlichting,I.&Weik,M.(2018),Naturechemistry,10,31–37.

Authors/affiliations:

De Zitter, E.¹*, Coquelle, N.², Barends, T.³, Colletier, J.-P^1#

¹Institut de Biologie Structurale(IBS), Univ.Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.

²Institut Laue-Langevin, 38044 Grenoble,France

³Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.