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Nucleation of an Activating Conformational Change by a Cation-# Interaction Per Rogne, David C. Andersson, Christin Grundström, A. Elisabeth Sauer-Eriksson, Anna Linusson, and Magnus Wolf-Watz Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00538 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019
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Biochemistry
Nucleation of an Activating Conformational Change by a Cation-π Interaction Per Rogne, David Andersson, Christin Grundström, Elisabeth Sauer-Eriksson, Anna Linusson and Magnus Wolf-Watz* Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden Supporting Information Available ABSTRACT: As a key molecule in biology, adenosine triphosphate (ATP) has numerous crucial functions in, for instance, energetics, post-translational modifications, nucleotide biosynthesis, and co-factor metabolism. Here, we have discovered an intricate interplay between the enzyme adenylate kinase and its substrate ATP. The side-chain of an arginine residue was found to be an efficient sensor of the aromatic moiety of ATP through the formation of a strong cation-π interaction. In addition to recognition, the interaction was found to have dual functionality. Firstly, it nucleates the activating conformational transition of the ATP binding domain and secondly, also affects the specificity in the distant AMP binding domain. In light of the functional consequences resulting from the cation-π interaction, it is possible that the mode of ATP recognition may be a useful tool in enzyme design.
Precise recognition and coordination of adenosine tri-phosphate (ATP) by proteins is of paramount importance for a large collection of biological processes, including transport1, signaling2, energy homeostasis3, molecular motors4, cofactor synthesis5, phosphorylation6 and nucleic acid metabolism6. Principles of ATP recognition mostly include interactions with the polar groups of ATP7, 8. We have previously discovered that ATP is selectively recognized by the metabolic enzyme adenylate kinase (Adk), a member of the NMP kinase family, through a hydrogen bond formed between the backbone carbonyl oxygen of lysine 200 (Lys200) and the NH2 group on carbon C6 of ATP9. This principle of ATP recognition, which is conserved in the family of eukaryotic protein kinases9, 10, enables Adk to discriminate between ATP and the chemically related molecule guanosine tri-phosphate (GTP)9. In search for additional principles in ATP recognition, examination of the molecular structure of closed and inhibitor bound Adk (1AKE.pdb)11 revealed that the sidechain of arginine 119 (Arg119) stacks against the adenosine base of ATP and likely contributes binding energy through a cation-π interaction (Figure 1A). A systematic analysis of crystal structures the NMP kinase family reveals that the equivalent of Arg119 is structurally conserved (Table S1 & Figure S1). In all instances were co-crystal structures were solved with substrates or inhibitors a corresponding putative cation-π interaction was observed. Here, we provide both experimental observations and computational results that demonstrates the importance of an enzyme to ATP cation-π interaction for proper function of Adk. The strategy was to perturb the wild-type enzyme by either making a conservative replacement of Arg119 for lysine (R119K) or by making an “alanine-scan” type of replacement of Arg119 (R119A). Results from both perturbations, revealed that the cation-π
Figure 1. Structure and functional consequence of the cation-π mediated ATP recognition by Adk. (A) Recognition of the adenosine base of ATP by a cation-π interaction donated from Arg119 in Adk. The sidechain of Arg119 is shown together with its surface envelope (orange) for closed Adk in complex with Ap5A (1AKE.pdb11). The ATPlid is shown in blue, the AMPbd is shown in dark green and alpha helix 1 is highlighted in yellow. (B) Schematic illustration of the dual functionalities of the cation-π interaction. Firstly, the interaction redistributes the ATPlid to predominantly populate the closed and active conformation placing the catalytically important residues Arg123 and Arg156 in close proximity with the phosphate groups of ATP. Secondly, the substrate specificity in the AMPbd is governed by an occlusion effect that relies on closure of the ATPlid. The hydrogen bond that is responsible for ATP vs GTP selectivity in Adk is shown as a dotted line9 (C) Zoom in on the sidechain of Arg119 and of the adenosine base corresponding to the ATP moiety of Ap5A (1AKE.pdb). (D) Structure of the bi-substrate inhibitor Ap5A.
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interaction is required for successful formation of the catalytically competent and closed Adk (Figure 1A). The binding free energy that is harnessed by the interaction is therefore, in part, used to funnel the ensemble of ATP binding domain (ATPlid) conformers towards the closed conformation (Figure 1B). Thus, there exist two crucial interactions with the adenine base of ATP, which are necessary to obtain the active conformation of Adk. First, and as demonstrated previously 9, the NH2 group of C6 is specifically recognized by the hydrogen bond donated to Lys200. Second, and as described here, the cationic sidechain of Arg119 that partitions the ATPlid conformers towards the closed state to enable catalysis. The cation-π interaction is not specific in the context of ATP versus GTP discrimination but is an efficient sensor for a spatially correctly placed aromatic system in ATP. In addition, we here found that the cation-π mediated closure of the ATPlid is crucial for AMP specificity in the spatially distant AMP binding domain (AMPbd) (Figures 1A and B). In the following sections, we describe the experiments that enabled these conclusions.
Figure 2. Redistribution of ATPlid conformers in response to the cation-π interaction. Closure of the ATPlid was monitored by quantifying line-broadening effects following TEMPO conjugation to position 148 in the presence of Ap5A. (A) Normalized peak intensities (Ipara/Idia) under conditions with saturating Ap5A concentrations are displayed against primary sequence for wild type Adk. The residues are clustered into groups with peak intensities within +/- 3 standard deviations (STD) from an average calculated for residues not affected by the PRE probe (blue; no effect), less than 3 STD below the average (yellow; moderate effect) and broadened beyond detection limit (red; strong effect). (B) Display of peak intensities for (wild type Adk on the open Adk structure (4AKE.pdb). The open structure is used for clarity and the color coding is as in (A)). (C) Normalized peak intensities with saturating Ap5A concentrations are displayed against primary sequence for R119A. (D) Display of peak intensities for R119A on the open Adk structure (4AKE.pdb). The color coding in (C) and (D) is the same as described for (A). Domain distances in proteins can be quantified with paramagnetic relaxation enhancement (PRE) experiments12. In this approach, a probe with an unpaired electron is attached to a protein and spatial proximity to the probe will cause increased transverse (R2) relaxation rates of observed NMR resonances. Proximity to the PRE probe can be detected as increased resonance linewidths and decreased peak-intensities that both are consequences of the increased R2 relaxation rates. Closure of the ATPlid in Adk was monitored by analyzing NMR resonance broadening following introduction of the paramagnetic spin-label TEMPO13 at position Val148 in the top of the ATPlid9. It has previously been established
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that the most sensitive marker for the closure event are residues in alpha helix one (residues 13-24 and colored yellow in Figure 1A). Here, we use the bi-substrate mimic Ap5A (P1,P5-Di(adenosine5')pentaphosphate) to stabilize the fully closed state of wild-type Adk (Figure 1A)14, 15. Ap5A is in essence an ATP and an AMP molecule bridged with a phosphodiester bond (Figure 1D). A comparison of paramagnetic broadening in Ap5A-saturated wildtype Adk and R119A revealed that the effects on alpha helix one are significantly smaller for R119A compared to the wild-type (Figure 2). These observations demonstrates that the ensemble of ATPlid conformers is biased towards the closed state for the wildtype but biased towards the open state for R119A. Thus, the chemical shifts of the Ap5A saturated R119A 1H-15N HSQC spectrum is reporting on an Adk state with an ATPlid that is biased towards the open state. Given that 1H-15N HSQC spectra of R119A and R119K saturated with Ap5A are very similar as quantified with linear correlations of chemical shifts (Figure S2) it can be deduced that the ATPlid in the R119K variant is also biased towards the open state in complex with Ap5A. Since both catalysis and binding affinities are dependent on closure of the ATPlid, they should be affected in R119A and R119K. Activities quantified with an ATPase assay16 showed that R119A only retained residual catalytic activity and that a modest rescue of activity was observed for the R119K variant. (Table 1). Any adverse effects on the structural integrity of both the R119K and R119A substitution were excluded on basis of NMR spectra and high-resolution crystallographic structures of apo R119K and R119A (Figure S3 and Table S2). Before crystallization Ap5A was added to the substituted variants proteins, however no Ap5A binding was observed and the mutant structures were in the substrate free open form. Hence, the reduced activities of the Adk variants is supporting a model were the ATPlid is predominantly in an open state in the R119A/K‒ATP complexes. A 17-fold increase in KM for the R119A‒ATP complex compared to the wild- type (Table 1) suggests that the binding affinity of R119A for ATP was reduced. The effect on KM for R119K is smaller (6-fold increase compared to the wild-type, Table 1). In light of the low activity of the R119K variant it appears that a productive Michaelis complex cannot be formed with this variant which is consistent with impaired ATPlid closure from 1H-15N HSQC spectra. Binding of ATP to Adk is a coupled process where the initial association event is followed by closure of the ATPlid over pre-bound ATP. The binding model predicts a decreased binding affinity for ATP to R119A and R119K since the step with ATPlid closure is virtually removed for these variants. Binding affinities (Kd values) for the two variants were quantified with NMR spectroscopy and benchmarked against values quantified earlier for the wild-type. Ap5A binds to Adk with a Kd of 140 nM17, 18 and occurs in the slow-exchange regime in NMR titrations (Figure 3A). Slow exchange is manifested as disappearance and reappearance of NMR resonances as the system is shifted from free to fully bound enzyme. For R119A, the affinity for Ap5A is increased to the extent that the binding regime is shifted from slow to fast on the NMR chemical shift time-scale (Figures 3A, B). Thus, resonances gradually move as the Ap5A concentration is increased and a fit to the resulting binding curve provides a Kd of 14 µM (Table 1). The binding experiments support a scenario where
Table 1, Quantification of catalysis and binding for wildtype, R119A, and R119K Adk WT
R119A
R119K
kcat
330 s-1
0.5 s-1
6.7 s-1
KM (ATP)
63 µM
1100 µM
398 µM
Kd (ATP)
50 µM
388 µM
103 µM
Kd (Ap5A)
0.14 µM
14 µM
0.58 µM
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Biochemistry used density functional theory (DFT) calculations to estimate the cation-π interaction energy 20, 21.
Figure 4. Distance dependency of the cation-π interaction energy based on DFT calculations. (A) Atoms for which interaction energy calculations were performed with the Arg119 side chain in orange and the adenine of ATP in green (1AKE.pdb). The dashed line indicates the distance between the two moieties. (B) Distance Figure 3. (A, B) Change of exchange behavior on the NMR chemical shift time-scale upon removal of the cation-π interaction. (A) Amino acid residue 73 shows a typical slow exchange pattern upon Ap5A titration to the wild-type Adk, the peak representing the apo state disappears and the peak representing the bound state appears with increasing substrate concentration. On the other hand, during titration of Ap5A to R119A (B) the peak position of residue 73 represents a weighted average of the apo and Ap5A bound states, typical for fast exchange. (C, D) Change of primary ATP binding site of Adk for R119A. Removal of the cation-π interaction and the resulting weakening of the ATP binding affinity to the ATP-site transforms the endogenous AMP binding site to the primary ATP binding site. (C) Residue specific and logarithmic chemical shift responses of ATP binding to R119A normalized with equivalent responses for the wild-type. Color coding is as follows; red- larger chemical shift difference for R119A, bluesmaller larger chemical shift difference for R119A and green- no difference between R119A and wild-type Adk. (D) A display of the residue specific responses to ATP binding for R119A on the open Adk structure shows that the primary ATP binding site for this Adk variant has shifted to the endogenous AMP binding site. the affinity for Ap5A is reduced in R119A due to the lack of the cation-π interaction which, in turn, leads to a partitioning of the ATPlid towards the open state. The lack of ATPlid closure is corroborated by the significant decrease in magnitudes of chemical shifts induced in the ATPlid of R119A and R119K following ATP binding (Figures 3C and S4). On the other hand, the chemical shift perturbations in the AMP binding domain (AMPbd) are significantly larger for the two variants compared to the wild type (Figures 3 and S4), suggesting that the primary ATP binding site of R119A and R119K has shifted from the endogenous ATP binding site to that of the AMP binding site. The wild-type enzyme does not bind ATP to the AMP binding site as inferred from the lack of ATP inhibition in activity assays19. Thus, the selectivity in the AMP site of the wild-type enzyme is dependent on an allosteric effect where correct coordination of ATP with a closed ATPlid occludes ATP from access to the AMP binding site (Figure 1B). An interrogation of the PDB database indicates that cation-π interactions are used across enzyme families in ATP recognition (Table S3 and Figure S5). Considering the dramatic influence on the conformational equilibrium by the cation-π interaction between Arg119 and the base of ATP and the prevalence of such interactions in other systems, this mode of recognition of aromatic substrates may be a tractable building block in enzyme design efforts. We
scan of the Arg119-adenine interaction energy in water, where the distance and corresponding energy of the closed(1AKE.pdb) and open form (4AKE.pdb) of Adk are indicated. Building on the geometry of the closed structure of the Adk‒Ap5A complex (Figures 4A and S6), we found that the binding energy in water for the cation-π interaction was -11.2 kJ mol-1, which is in good agreement with the experimental estimate of the cation-π interaction (-11.5 kJ mol-1) calculated from the difference in binding energy for Ap5A comparing wild-type, and R119A Adk (Table 1). Notably, calculations of the interactions between the mutant R119K and the adenine moiety showed that thie interaction energy is virtually absent in water (Table S4 and Figure S6), indicating that the interaction energy between lysine and the base of ATP is very weak, corroborating the experimental conclusion. Next, we investigated the distance dependency of the Arg119 and ATP cation- interaction on a spatial trajectory equivalent to closing of the ATPlid over a pre-bound ATP molecule (Figures 4B, S6 and S7)22. There exist a significant interaction (-6.1 kJ mol-1) between Arg119 and ATP in the open Adk conformation, suggesting that pre-bound ATP exerts an attractive force on Arg119 with the ATPlid in the open conformation. Therefore, it appears that full closure of the ATPlid in the presence of pre-bound ATP, at least in part, is nucleated by the cation-π interaction. It has been shown previously that Adk has the innate capacity to close the ATPlid even in the absence of substrate23, 24 and that the rate constant for closing is increased approximately two-fold in the presence of AMP-PNP relative to the substrate free scenario25. The observed increase in the rate-constant for closing in presence of an ATP analogue is consistent with an attractive nature of the cationπ interaction. Taken together an ATP binding model with properties from both induced fit26 and conformational selection27 models is emerging. As mentioned, Adk has an innate property that allows sampling of the catalytically competent and active state in the absence of substrate (conformational selection property). In the presence of bound ATP the partitioning between open and closed ATPlid conformations is funneled towards the closed conformation through thermal fluctuations (conformational selection property) that are reinforced by the attractive force (induced fit property) of the cation-π interaction. The cation-π interaction is specific towards aromatic substrates and we speculate that the binding mode could be used (possibly together with recent advancements in the design of p-loop sequences28) to support developments in rational design of enzymes utilizing ATP for their functionality.
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ASSOCIATED CONTENT Supporting Information Supporting Tables, figures, and experimental methods (PDF)
Accession codes Protein data bank entries: 6RZE (R119A) and 6S36 (R119K) BMRB entries: 26999 (WT Apo form), 27004 (WT Ap5A bound), 27005 (WT ATP bound), 27000 (R119A Apo form), 27007 (R119A Ap5A bound), 27008 (R119A ATP bound), 27001 (R119K Apo form), 27010 (R119K Ap5A bound), and 27954 (R119K ATP bound). The Uniprot Accession ID for Adenylate kinase from E. coli is P69441.
AUTHOR INFORMATION Corresponding Author *
[email protected] Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was financially supported by the Swedish Research Council to M.W.-W (04203) and A.L (05176). The authors would like to thank The Swedish National Infrastructure for Computing (SNIC) and the High Performance Computing Center North (HPC2N) for calculation resources. NMR experiments were performed at the Swedish NMR center “NMR for life” at Umeå University.
REFERENCES (1) Andersson, M.; Mattle, D.; Sitsel, O.; Klymchuk, T.; Nielsen, A. M.; Moller, L. B.; White, S. H.; Nissen, P.; Gourdon, P., Copper-transporting P-type ATPases use a unique ion-release pathway. Nat Struct Mol Bio 2014, 21 (1), 43-48. (2) Shah, N. H.; Kuriyan, J., Understanding molecular mechanisms in cell signaling through natural and artificial sequence variation. Nat Struct Mol Bio 2019, 26 (1), 25-34. (3) Tukenmez, H.; Magnussen, H. M.; Kovermann, M.; Byström, A.; WolfWatz, M., Linkage between fitness of Yeast cells and adenylate kinase catalysis. Plos One 2016, 11 (9), 15. No. e0163115 (4) Wulf, S. F.; Ropars, V.; Fujita-Becker, S.; Oster, M.; Hofhaus, G.; Trabuco, L. G.; Pylypenko, O.; Sweeney, H. L.; Houdusse, A. M.; Schroder, R. R., Force-producing ADP state of myosin bound to actin. Proc Nat Acad Sci USA 2016, 113 (13), E1844-E1852. (5) Bauer, S.; Kemter, K.; Bacher, A.; Huber, R.; Fischer, M.; Steinbacher, S., Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold. J Mol Biol 2003, 326 (5), 1463-1473. (6) Berg, J. M.; Tymoczko, J. L.; Stryer, L., Biochemistry. Fifth ed.; W.H. Freeman and Company: New York, USA, 2002. (7) Huse, M.; Kuriyan, J., The conformational plasticity of protein kinases. Cell 2002, 109 (3), 275-282. (8) Walker, J. E.; Saraste, M.; Runswick, M. J.; Gay, N. J., Distantly related sequences in the alpha-subunits and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. Embo J 1982, 1 (8), 945-951. (9) Rogne, P.; Rosselin, M.; Grundström, C.; Hedberg, C.; Sauer, U. H.; Wolf-Watz, M., Molecular mechanism of ATP versus GTP selectivity of adenylate kinase. Proc Nat Aca Scie USA 2018, 115 (12), 3012-3017.
(10) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S., The protein kinase complement of the human genome. Science 2002, 298 (5600), 1912-1934. (11) Müller, C. W.; Schulz, G. E., Structure of the complex between adenylate kinase from Escherichia coli and the inhibitor AP5A refined at 1.9 A resolution - A model for a catalytic transition-State. J Mol Biol 1992, 224 (1), 159-177. (12) Tang, C.; Schwieters, C. D.; Clore, G. M., Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR. Nature 2007, 449 (7165), 1078-U12. (13) Kosen, P. A., Spin labeling of proteins. Methods Enzymol 1989, 177, 86-121 (14) Lienhard, G. E.; Secemski, I. I., P1,P5-di(adenosine5')pentaphosphate, a Potent multisubstrate inhibitor of adenylate kinase. J Biol Chem 1973, 248 (3), 1121-1123. (15) Åden, J.; Wolf-Watz, M., NMR identification of transient complexes critical to adenylate kinase catalysis. J Am Chem Soc 2007, 129 (45), 1400314012. (16) Rhoads, D. G.; Lowenstein, J. M., Initial velocity and equilibrium kinetics of myokinase. J Biol Chem 1968, 243 (14), 3963-3972. (17) Schrank, T. P.; Bolen, D. W.; Hilser, V. J., Rational modulation of conformational fluctuations in adenylate kinase reveals a local unfolding mechanism for allostery and functional adaptation in proteins. Proc Nat Acad Sci USA 2009, 106 (40), 16984-16989. (18) Olsson, U.; Wolf-Watz, M., Overlap between folding and functional energy landscapes for adenylate kinase conformational change. Nat Commun 2010, 1, 111. Artn. 111. (19) Åden, J.; Verma, A.; Schug, A.; Wolf-Watz, M., Modulation of a preexisting conformational equilibrium tunes adenylate kinase activity. J Am Chem Soc 2012, 134 (40), 16562-16570. (20) Kumar, K.; Woo, S. M.; Siu, T.; Cortopassi, W. A.; Duarte, F.; Paton, R. S., Cation-pi interactions in protein-ligand binding: theory and datamining reveal different roles for lysine and arginine. Chem Sci 2018, 9 (10), 2655-2665. (21) Cortopassi, W. A.; Kumar, K.; Paton, R. S., Cation-pi interactions in CREBBP bromodomain inhibition: an electrostatic model for smallmolecule binding affinity and selectivity. Org Biomol Chem 2016, 14 (46), 10926-10938. (22) Ådén, J.; Weise, C. F.; Brännström, K.; Olofsson, A.; Wolf-Watz, M., Structural topology and activation of an initial adenylate kinase–substrate complex. Biochemistry 2013, 52 (6), 1055-1061. (23) Kovermann, M.; Grundström, C.; Sauer-Eriksson, A. E.; Sauer, U. H.; Wolf-Watz, M., Structural basis for ligand binding to an enzyme by a conformational selection pathway. Proc Nat Aca Sci USA 2017, 114 (24), 6298-6303. (24) Henzler-Wildman, K. A.; Thai, V.; Lei, M.; Ott, M.; Wolf-Watz, M.; Fenn, T.; Pozharski, E.; Wilson, M. A.; Petsko, G. A.; Karplus, M.; Hubner, C. G.; Kern, D., Intrinsic motions along an enzymatic reaction trajectory. Nature 2007, 450 (7171), 838-844. (25) Hanson, J. A.; Duderstadt, K.; Watkins, L. P.; Bhattacharyya, S.; Brokaw, J.; Chu, J.-W.; Yang, H., Illuminating the mechanistic roles of enzyme conformational dynamics. Proc Nat Aca Sci USA 2007, 104 (46), 18055-18060. (26) Koshland, D. E., Application of a theory of enzyme specificity to protein synthesis. Proc Nat Aca Sci USA 1958, 44 (2), 98-104. (27) Lange, O. F.; Lakomek, N. A.; Fares, C.; Schroder, G. F.; Walter, K. F. A.; Becker, S.; Meiler, J.; Grubmuller, H.; Griesinger, C.; de Groot, B. L., Recognition dynamics up to microseconds revealed from an RDCderived ubiquitin ensemble in solution. Science 2008, 320 (5882), 14711475. (28) Romero, M. L. R.; Yang, F.; Lin, Y. R.; Toth-Petroczy, A.; Berezovsky, I. N.; Goncearenco, A.; Yang, W.; Wellner, A.; KumarDeshmukh, F.; Sharon, M.; Baker, D.; Varani, G.; Tawfik, D. S., Simple yet functional phosphate-loop proteins. Proc Nat Aca Sci USA 2018, 115 (51), E11943-E11950.
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