Predicting protein conformational motions using energetic frustration analysis and AlphaFold2.

Proteins perform their biological functions through motion. Although high throughput prediction of the three-dimensional static structures of proteins has proved feasible using deep-learning-based methods, predicting the conformational motions remains a challenge. Purely data-driven machine learning methods encounter difficulty for addressing such motions because available laboratory data on conformational motions are still limited. In this work, we develop a method for generating protein allosteric motions by integrating physical energy landscape information into deep-learning-based methods. We show that local energetic frustration, which represents a quantification of the local features of the energy landscape governing protein allosteric dynamics, can be utilized to empower AlphaFold2 (AF2) to predict protein conformational motions. Starting from ground state static structures, this integrative method generates alternative structures as well as pathways of protein conformational motions, using a progressive enhancement of the energetic frustration features in the input multiple sequence alignment sequences. For a model protein adenylate kinase, we show that the generated conformational motions are consistent with available experimental and molecular dynamics simulation data. Applying the method to another two proteins KaiB and ribose-binding protein, which involve large-amplitude conformational changes, can also successfully generate the alternative conformations. We also show how to extract overall features of the AF2 energy landscape topography, which has been considered by many to be black box. Incorporating physical knowledge into deep-learning-based structure prediction algorithms provides a useful strategy to address the challenges of dynamic structure prediction of allosteric proteins.

The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation.

Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNA⋅tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.

The Biochemical Impact of Extracting an Embedded Adenylate Kinase Domain Using Circular Permutation.

Adenylate kinases (AKs) have evolved AMP-binding and lid domains that are encoded as continuous polypeptides embedded at different locations within the discontinuous polypeptide encoding the core domain. A prior study showed that AK homologues of different stabilities consistently retain cellular activity following circular permutation that splits a region with high energetic frustration within the AMP-binding domain into discontinuous fragments. Herein, we show that mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologues at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30 °C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than those of native AKs, and they exhibited multiphasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK in differing manners, and mass spectrometry suggested that the new termini within the AMP-binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes.

Elucidating Dynamics of Adenylate Kinase from Enzyme Opening to Ligand Release.

This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.

Dynamic allostery can drive cold adaptation in enzymes.

Adaptation of organisms to environmental niches is a hallmark of evolution. One prevalent example is that of thermal adaptation, in which two descendants evolve at different temperature extremes1,2. Underlying the physiological differences between such organisms are changes in enzymes that catalyse essential reactions 3 , with orthologues from each organism undergoing adaptive mutations that preserve similar catalytic rates at their respective physiological temperatures4,5. The sequence changes responsible for these adaptive differences, however, are often at surface-exposed sites distant from the substrate-binding site, leaving the active site of the enzyme structurally unperturbed6,7. How such changes are allosterically propagated to the active site, to modulate activity, is not known. Here we show that entropy-tuning changes can be engineered into distal sites of Escherichia coli adenylate kinase, allowing us to quantitatively assess the role of dynamics in determining affinity, turnover and the role in driving adaptation. The results not only reveal a dynamics-based allosteric tuning mechanism, but also uncover a spatial separation of the control of key enzymatic parameters. Fluctuations in one mobile domain (the LID) control substrate affinity, whereas dynamic attenuation in the other domain (the AMP-binding domain) affects rate-limiting conformational changes that govern enzyme turnover. Dynamics-based regulation may thus represent an elegant, widespread and previously unrealized evolutionary adaptation mechanism that fine-tunes biological function without altering the ground state structure. Furthermore, because rigid-body conformational changes in both domains were thought to be rate limiting for turnover8,9, these adaptation studies reveal a new model for understanding the relationship between dynamics and turnover in adenylate kinase.

Insights into Enzymatic Catalysis from Binding and Hydrolysis of Diadenosine Tetraphosphate by E. coli Adenylate Kinase.

Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.

An open interface in the pre-80S ribosome coordinated by ribosome assembly factors Tsr1 and Dim1 enables temporal regulation of Fap7.

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved methyltransferase Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality control during ribosome assembly.

Towards the energy landscape of adenylate kinase in crowded milieu: Activity, conformation, structure and dynamics in sequence.

Enzyme function is governed by a complex network of conformational changes and internal dynamics, with the same getting more convoluted in the crowded cellular environment. Here, we have explored an intricate interplay amongst activity, structure, conformation, and dynamics of a multidomain enzyme, AK3L1 (UniProtKB: Q9UIJ7) in the crowded milieu. We have monitored changes in the enzyme landscape in response to the chemical denaturant, urea, under the influence of different concentrations of macromolecular crowders. Extensive experimental analyses using FRET-based domain displacement measurements, sub-nanosecond time scale local dynamics, and global structural changes, along with enzymatic activity studies, have been carried out to get deeper insights into the factors that may modulate the functional landscape of adenylate kinase (AK3L1). It was observed that AK3L1 gets activated at low urea concentrations, whereas higher urea concentrations unfold and thereby deactivate the enzyme. A sequential response of AK3L1 is observed towards external perturbation (urea) occurring through a series of well-defined steps. Incorporation of crowders not only shift the maximum activity of enzyme to a higher urea concentration, but also enhance domain compaction, as revealed by FRET studies. The modulation in enzyme activity and solvation dynamics acting as local response, precede global unfolding of the enzyme, indicating that the structural alterations around the active site are quite decoupled from the large amplitude global transitions.

Adenylate Kinase Fused to Spidroin as a Catalyst for Decreasing Leakage out of 3D-Bioprinted Hydrogels and for ATP Regeneration.

Numerous metabolic reactions and pathways use adenosine 5'-triphosphate (ATP) as an energy source and as a phosphorous or pyrophosphorous donor. Based on three-dimensional (3D)-printing, enzyme immobilization can be used to improve ATP regeneration and operability and reduce cost. However, due to the relatively large mesh size of 3D-bioprinted hydrogels soaked in a reaction solution, the lower-molecular-weight enzymes cannot avoid leaking out of the hydrogels readily. Here, a chimeric adenylate-kinase-spidroin (ADK-RC) is created, with ADK serving as the N-terminal domain. The chimera is capable of self-assembling to form micellar nanoparticles at a higher molecular scale. Although fused to spidroin (RC), ADK-RC remains relatively consistent and exhibits high activity, thermostability, pH stability, and organic solvent tolerance. Considering different surface-to-volume ratios, three shapes of enzyme hydrogels are designed, 3D bioprinted, and measured. In addition, a continuous enzymatic reaction demonstrates that ADK-RC hydrogels have higher specific activity and substrate affinity but a lower reaction rate and catalytic power compared to free enzymes in solution. With ATP regeneration, the ADK and ADK-RC hydrogels significantly increase the production of d-glucose-6-phosphate and obtain an efficient usage frequency. In conclusion, enzymes fused to spidroin might be an efficient strategy for maintaining activity and reducing leakage in 3D-bioprinted hydrogels under mild conditions.

Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase.

Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme's conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme's function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK's conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme's conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme's active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.

Moving beyond static snapshots: Protein dynamics and the Protein Data Bank.

Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions. Although the dynamics are more difficult to observe than a static, average structure, we are beginning to observe these dynamics and form sound mechanistic connections between structure, dynamics, and function. This progress is highlighted in case studies from myoglobin and adenylate kinase to the ribosome and molecular motors where these molecules are being probed with a multitude of techniques across many timescales. New approaches to time-resolved crystallography are allowing simple "movies" to be taken of proteins in action, and new methods of mapping the variations in cryo-electron microscopy are emerging to reveal a more complete description of life's machines. The results of these new methods are aided in their dissemination by continual improvements in curation and distribution by the Protein Data Bank and their partners around the world.

Integration of Adenylate Kinase 1 with Its Peptide Conformational Imprint.

In the present study, molecularly imprinted polymers (MIPs) were used as a tool to grasp a targeted α-helix or ß-sheet of protein. During the fabrication of the hinge-mediated MIPs, elegant cavities took shape in a special solvent on quartz crystal microbalance (QCM) chips. The cavities, which were complementary to the protein secondary structure, acted as a peptide conformational imprint (PCI) for adenylate kinase 1 (AK1). We established a promising strategy to examine the binding affinities of human AK1 in conformational dynamics using the peptide-imprinting method. Moreover, when bound to AK1, PCIs are able to gain stability and tend to maintain higher catalytic activities than free AK1. Such designed fixations not only act on hinges as accelerators; some are also inhibitors. One example of PCI inhibition of AK1 catalytic activity takes place when PCI integrates with an AK19-23 ß-sheet. In addition, conformation ties, a general MIP method derived from random-coil AK1133-144 in buffer/acetonitrile, are also inhibitors. The inhibition may be due to the need for this peptide to execute conformational transition during catalysis.

Dynamic Connection between Enzymatic Catalysis and Collective Protein Motions.

Enzymes employ a wide range of protein motions to achieve efficient catalysis of chemical reactions. While the role of collective protein motions in substrate binding, product release, and regulation of enzymatic activity is generally understood, their roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the roles of catalytic residues in catalysis and the conformational change in the enzyme. This study reveals that the motions in the active site, which occur on a time scale of picoseconds to nanoseconds, link the catalytic reaction to the slow conformational dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In particular, substantial conformational rearrangement occurs in the active site following the catalytic reaction. This rearrangement not only affects the reaction barrier but also promotes a more open conformation of the enzyme after the reaction, which then results in an accelerated opening of the enzyme compared to that of the reactant state. The results illustrate a linkage between enzymatic catalysis and collective protein motions, whereby the disparate time scales between the two processes are bridged by a cascade of intermediate-scale motion of catalytic residues modulating the free energy landscapes of the catalytic and conformational change processes.

Adenylate kinases of thermophiles Aquifex aeolicus and Geobacillus stearothermophilus: biochemical and kinetic studies.

Adenylate kinases (AK) play a pivotal role in the regulation of cellular energy. The aim of our work was to achieve the overproduction and purification of AKs from two groups of bacteria and to determine, for the first time, the comprehensive biochemical and kinetic properties of adenylate kinase from Gram-negative Aquifex aeolicus (AKaq) and Gram-positive Geobacillus stearothermophilus (AKst). Therefore we determined KM and Vmax values, and the effects of temperature, pH, metal ions, donors of the phosphate groups and inhibitor Ap5A for both thermophilic AKs. The kinetic studies indicate that both AKs exhibit significantly higher affinity for substrates with the pyrophosphate group than for adenosine monophosphate. AK activation by Mg2+ and Mn2+ revealed that both ions are efficient in the synthesis of adenosine diphosphate and adenosine triphosphate; however, Mn2+ ions at 0.2-2.0 mmol/L concentration were more efficient in the activation of the ATP synthesis than Mg2+ ions. Our research demonstrates that zinc ions inhibit the activity of enzymes in both directions, while Ap5A at a concentration of 10 µmol/L and 50 µmol/L inhibited both enzymes with a different efficiency. Sigmoid-like kinetics were detected at high ATP concentrations not balanced by Mg2+, suggesting the allosteric effect of ATP for both bacterial AKs.

Impacts of mutations on dynamic allostery of adenylate kinase.

Escherichia coli adenylate kinase (AK) is composed of CORE domain and two branch domains: LID and AMP-binding domain (AMPbd). AK exhibits considerable allostery in a reversible phosphoryl transfer reaction, which is largely attributed to the relative motion of LID and AMPbd with respect to CORE. Such an allosteric conformational change is also evident in the absence of ligands. Recent studies showed that the mutations in branch domains can adjust dynamic allostery and alter the substrate affinity and enzyme activity. In this work, we use all-atom molecular dynamics simulation to study the impacts of mutations in branch domains on AK's dynamic allostery by comparing two double mutants, i.e., LID mutant (Val135Gly, Val142Gly) and AMPbd mutant (Ala37Gly, Ala55Gly), with wild-type. Two mutants undergo considerable conformational fluctuation and exhibit deviation from the initially extended apo state to more compact structures. The LID domain in the LID mutant adjusts its relative position and firmly adheres to CORE. More strikingly, AMPbd mutations affect the relative positions of both the AMPbd domain and remote LID domain. Both domains undergo considerable movement, especially the inherent hinge swing motion of the flexible LID domain. In both mutants, the mutations can enhance the inter-domain interaction. The results about the conformation change of AK in both mutants are in line with the experiment of AK's affinity and activity. As revealed by our findings, the flexibility of branch domains and their inherent motions, especially LID domain, is highly relevant to dynamic allostery in the AK system.

Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions.

The functional cycle of many proteins involves large-scale motions of domains and subunits. The relation between conformational dynamics and the chemical steps of enzymes remains under debate. Here we show that in the presence of substrates, domain motions of an enzyme can take place on the microsecond time scale, yet exert influence on the much-slower chemical step. We study the domain closure reaction of the enzyme adenylate kinase from Escherichia coli while in action (i.e., under turnover conditions), using single-molecule FRET spectroscopy. We find that substrate binding increases dramatically domain closing and opening times, making them as short as ∼15 and ∼45 µs, respectively. These large-scale conformational dynamics are likely the fastest measured to date, and are ∼100-200 times faster than the enzymatic turnover rate. Some active-site mutants are shown to fully or partially prevent the substrate-induced increase in domain closure times, while at the same time they also reduce enzymatic activity, establishing a clear connection between the two phenomena, despite their disparate time scales. Based on these surprising observations, we propose a paradigm for the mode of action of enzymes, in which numerous cycles of conformational rearrangement are required to find a mutual orientation of substrates that is optimal for the chemical reaction.

Rational Design of Adenylate Kinase Thermostability through Coevolution and Sequence Divergence Analysis.

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature.

Assessing the Interactions of Statins with Human Adenylate Kinase Isoenzyme 1: Fluorescence and Enzyme Kinetic Studies.

Statins are the most effective cholesterol-lowering drugs. They also exert many pleiotropic effects, including anti-cancer and cardio- and neuro-protective. Numerous nano-sized drug delivery systems were developed to enhance the therapeutic potential of statins. Studies on possible interactions between statins and human proteins could provide a deeper insight into the pleiotropic and adverse effects of these drugs. Adenylate kinase (AK) was found to regulate HDL endocytosis, cellular metabolism, cardiovascular function and neurodegeneration. In this work, we investigated interactions between human adenylate kinase isoenzyme 1 (hAK1) and atorvastatin (AVS), fluvastatin (FVS), pravastatin (PVS), rosuvastatin (RVS) and simvastatin (SVS) with fluorescence spectroscopy. The tested statins quenched the intrinsic fluorescence of hAK1 by creating stable hAK1-statin complexes with the binding constants of the order of 104 M-1. The enzyme kinetic studies revealed that statins inhibited hAK1 with significantly different efficiencies, in a noncompetitive manner. Simvastatin inhibited hAK1 with the highest yield comparable to that reported for diadenosine pentaphosphate, the only known hAK1 inhibitor. The determined AK sensitivity to statins differed markedly between short and long type AKs, suggesting an essential role of the LID domain in the AK inhibition. Our studies might open new horizons for the development of new modulators of short type AKs.

Structural Basis for GTP versus ATP Selectivity in the NMP Kinase AK3.

ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3, which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle, where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and nonproductively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and nonproductive binding by the incorrect substrate bear a strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together, we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from nonlinearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These nonspecific interactions likely act to lower the available intracellular GTP and ATP concentrations and may have driven evolution of the Michaelis constants of NMP kinases accordingly.

Optical Control of Cellular ATP Levels with a Photocaged Adenylate Kinase.

We have developed a new tool for the optical control of cellular ATP concentrations with a photocaged adenylate kinase (Adk). The photocaged Adk is generated by substituting a catalytically essential lysine with a hydroxycoumarin-protected lysine through site-specific unnatural amino acid mutagenesis in both E. coli and mammalian cells. Caging of the critical lysine residue offers complete suppression of Adk's phosphotransferase activity and rapid restoration of its function both in vitro and in vivo upon optical stimulation. Light-activated Adk renders faster rescue of cell growth than chemically inducible expression of wild-type Adk in E. coli as well as rapid ATP depletion in mammalian cells. Thus, caging Adk provides a new tool for direct conditional perturbation of cellular ATP concentrations thereby enabling the investigation of ATP-coupled physiological events in temporally dynamic contexts.