NMR Study Suggests a Major Role for Arg111 in Maintaining the Structure and Dynamical Properties of Type II Human Cellular Retinoic Acid Binding Protein^†

The solution structure of a site-directed mutant of type-II human cellular retinoic acid binding protein (CRABPII) with Arg111 replaced by methionine (R111M) has been determined by NMR spectroscopy. The sequential assignments of the 1H and 15N resonances of apo-R111M were established by multinuclear multidimensional NMR. The solution structure was calculated from 2302 distance restraints and 77 φ dihedral restraints derived from the NMR data. The root-mean-square deviation of the ensemble of 28 refined conformers that represent the structure from the mean coordinate set derived from them was 0.54 ± 0.26 and 0.98 ± 0.23 Å for the backbone atoms and all heavy atoms, respectively. The solution structure of apo-R111M is similar to that of wild-type apo-CRABPII. However, there are significant conformational differences between the two proteins, localized mainly to three segments (Leu19−Ala36, Glu73−Cys81, and Leu99−Pro105) clustered around the ligand entrance more than 17 Å away from the point mutation. In apo-R111M, all the three segments move toward the center of the ligand entrance so that the opening of the ligand-binding pocket in apo-R111M is much smaller than that in wild-type apo-CRABPII. Furthermore, the ligand-binding pocket of apo-R111M, especially the ligand entrance, is much less flexible than that of apo-CRABPII. Surprisingly, apo-R111M is more similar to holo-CRABPII than to apo-CRABPII in both structure and dynamical properties. The conformational and dynamical changes caused by the mutation are similar to those induced by binding of RA, although the magnitudes of the changes caused by the mutation are smaller than those induced by binding of RA. The results suggest that Arg111 plays a critical role in determining the structure and dynamical properties of CRABPII

NMR Study Suggests a Major Role for Arg111 in Maintaining the Structure and Dynamical Properties of Type II Human Cellular Retinoic Acid Binding Protein^†

The solution structure of a site-directed mutant of type-II human cellular retinoic acid binding protein (CRABPII) with Arg111 replaced by methionine (R111M) has been determined by NMR spectroscopy. The sequential assignments of the 1H and 15N resonances of apo-R111M were established by multinuclear multidimensional NMR. The solution structure was calculated from 2302 distance restraints and 77 φ dihedral restraints derived from the NMR data. The root-mean-square deviation of the ensemble of 28 refined conformers that represent the structure from the mean coordinate set derived from them was 0.54 ± 0.26 and 0.98 ± 0.23 Å for the backbone atoms and all heavy atoms, respectively. The solution structure of apo-R111M is similar to that of wild-type apo-CRABPII. However, there are significant conformational differences between the two proteins, localized mainly to three segments (Leu19−Ala36, Glu73−Cys81, and Leu99−Pro105) clustered around the ligand entrance more than 17 Å away from the point mutation. In apo-R111M, all the three segments move toward the center of the ligand entrance so that the opening of the ligand-binding pocket in apo-R111M is much smaller than that in wild-type apo-CRABPII. Furthermore, the ligand-binding pocket of apo-R111M, especially the ligand entrance, is much less flexible than that of apo-CRABPII. Surprisingly, apo-R111M is more similar to holo-CRABPII than to apo-CRABPII in both structure and dynamical properties. The conformational and dynamical changes caused by the mutation are similar to those induced by binding of RA, although the magnitudes of the changes caused by the mutation are smaller than those induced by binding of RA. The results suggest that Arg111 plays a critical role in determining the structure and dynamical properties of CRABPII

Conformational Transition in Signal Transduction: Metastable States and Transition Pathways in the Activation of a Signaling Protein

Signal transduction is of vital importance to the growth and adaptation of living organisms. The key to understand mechanisms of biological signal transduction is elucidation of the conformational dynamics of its signaling proteins, as the activation of a signaling protein is fundamentally a process of conformational transition from an inactive to an active state. A predominant form of signal transduction for bacterial sensing of environmental changes in the wild or inside their hosts is a variety of two-component systems, in which the conformational transition of a response regulator (RR) from an inactive to an active state initiates responses to the environmental changes. Here, RR activation has been investigated using RR468 as a model system by extensive unbiased all-atom molecular dynamics (MD) simulations in explicit solvent, starting from snapshots along a targeted MD trajectory that covers the conformational transition. Markov state modeling, transition path theory, and geometric analyses of the wealth of the MD data have provided a comprehensive description of the RR activation. It involves a network of metastable states, with one metastable state essentially the same as the inactive state and another very similar to the active state that are connected via a small set of intermediates. Five major pathways account for >75% of the fluxes of the conformational transition from the inactive to the active-like state. The thermodynamic stability of the states and the activation barriers between states are found, to identify rate-limiting steps. The conformal transition is initiated predominantly by movements of the β3α3 loop, followed by movements of the β4α4-loop and neighboring α4 helix region, and capped by additional movements of the β3α3 loop. A number of transient hydrophobic and hydrogen bond interactions are revealed, and they may be important for the conformational transition

Conformational Transition of Response Regulator RR468 in a Two-Component System Signal Transduction Process

Signal transduction can be accomplished via a two-component system (TCS) consisting of a histidine kinase (HK) and a response regulator (RR). In this work, we simulate the response regulator RR468 from Thermotoga maritima, in which phosphorylation and dephosphorylation of a conserved aspartate residue acts as a switch via a large conformational change concentrated in three proximal loops. A detailed view of the conformational transition is obscured by the lack of stability of the intermediate states, which are difficult to detect using common structural biology techniques. Molecular dynamics (MD) trajectories of the inactive and active conformations were run, and show that the inactive (or active) trajectories do not exhibit sampling of the active (or inactive) conformations on this time scale. Targeted MD (TMD) was used to generate trajectories that span the inactive and active conformations and provide a view of how a localized event like phosphorylation can lead to conformational changes elsewhere in the protein, especially in the three proximal loops. The TMD trajectories are clustered to identify stages along the transition path. Residue interaction networks are identified that point to key residues having to rearrange in the process of transition. These are identified using both hydrogen bond analysis and residue interaction strength measurements. Potentials of mean force are generated for key residue rearrangements to ascertain their free energy barriers. We introduce methods that attempt to extrapolate from one conformation to the other and find that the most fluctuating proximal loop can transit part way from one to the other, suggesting that this conformational information is embedded in the sequence

Dynamics in <i>Thermotoga neapolitana</i> Adenylate Kinase: ¹⁵N Relaxation and Hydrogen−Deuterium Exchange Studies of a Hyperthermophilic Enzyme Highly Active at 30 °C

Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 °C using 15N NMR relaxation measurements and NMR monitored hydrogen−deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 °C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 °C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core−lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK’s increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen−deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 °C by localizing flexibility to the hinge regions that are key to facilitating conformational changes

Bisubstrate Analogue Inhibitors of 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase: Synthesis and Biochemical and Crystallographic Studies^†

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. Like other enzymes in the folate pathway, HPPK is an ideal target for the development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from human and animals. Three bisubstrate analogues have been synthesized for HPPK and characterized by biochemical and X-ray crystallographic analyses. All three bisubstrate analogues consist of a pterin, an adenosine moiety, and a link composed of 2−4 phosphoryl groups. P-(6-Hydroxymethylpterin)-P-(5‘-adenosyl)diphosphate (HP2A, 5) shows little affinity and inhibitory activity for E. coli HPPK. P1-(6-Hydroxymethylpterin)-P-(5‘-adenosyl)triphosphate (HP3A, 6) shows moderate affinity and inhibitory activity with Kd = 4.25 μM in the presence of Mg2+ and IC50 = 1.27 μM. P1-(6-Hydroxymethylpterin)-P-(5‘-adenosyl)tetraphosphate (HP4A, 7) shows the highest affinity and inhibitory activity with Kd = 0.47 μM in the presence of Mg2+ and IC50 = 0.44 μM. The affinity of MgHP4A for HPPK is ∼116 and 76 times higher than that of MgADP and 6-hydroxymethylpterin, respectively. The crystal structure of HPPK in complex with 7 (HPPK·MgHP4A) has been determined at 1.85 Å resolution with a crystallographic R factor of 0.185. The crystal structure shows that 7 occupies both HP- and ATP-binding sites and induces significant conformational changes in HPPK. The biochemical and structural studies of the bisubstrate analogues indicate that the bisubstrate analogue approach can produce more potent inhibitors for HPPK and the minimum length of the link for a bisubstrate analogue is ∼7 Å

Mechanism of the Conformational Transitions in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by NMR Spectroscopy^†^,^‡

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. HPPK undergoes dramatic conformational changes during its catalytic cycle, and the conformational changes are essential for enzymatic catalysis. Thus, the enzyme is not only an attractive target for developing antimicrobial agents but also an excellent model system for studying the catalytic mechanism of enzymatic pyrophosphoryl transfer as well as the role of protein dynamics in enzymatic catalysis. In the present study, we report the NMR solution structures of the binary complex HPPK•MgAMPCPP and the ternary complex HPPK•MgAMPCPP•DMHP, where α,β-methyleneadenosine triphosphate (AMPCPP) and 7, 7-dimethyl-6-hydroxypterin (DMHP) are the analogues of the substrates ATP and HP, respectively. The results suggest that the three catalytic loops of the binary complex of HPPK can assume multiple conformations in slow exchanges as evidenced by multiple sets of NMR signals for several residues in loops 2 and 3 and the very weak or missing NH cross-peaks for several residues in loops 1 and 3. However, the ternary complex shows only one set of NMR signals, and the cross-peak intensities are rather uniform, suggesting that the binding of the second substrate shifts the multiple conformations of the binary complex to an apparently single conformation of the ternary complex. The NMR behaviors and conformations of the binary complex HPPK•MgAMPCPP are significantly different from those of HPPK in complex with Mgβ,γ-methyleneadenosine triphosphate (MgAMPPCP). It is suggested that the conformational properties of the binary substrate complex HPPK•MgATP be represented by those of HPPK•MgAMPCPP, because MgAMPCPP is a better MgATP analogue for HPPK with respect to both binding affinity and bound conformation

Dynamics in <i>Thermotoga neapolitana</i> Adenylate Kinase: ¹⁵N Relaxation and Hydrogen−Deuterium Exchange Studies of a Hyperthermophilic Enzyme Highly Active at 30 °C

Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 °C using 15N NMR relaxation measurements and NMR monitored hydrogen−deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 °C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 °C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core−lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK’s increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen−deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 °C by localizing flexibility to the hinge regions that are key to facilitating conformational changes

Molecular Dynamics Simulations of the <i>Escherichia coli</i> HPPK Apo-enzyme Reveal a Network of Conformational Transitions

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the first reaction in the folate biosynthetic pathway. Comparison of its X-ray and nuclear magnetic resonance structures suggests that the enzyme undergoes significant conformational change upon binding to its substrates, especially in three catalytic loops. Experimental research has shown that even when confined by crystal contacts, loops 2 and 3 remain rather flexible when the enzyme is in its apo form, raising questions about the putative large-scale induced-fit conformational change of HPPK. To investigate the loop dynamics in a crystal-free environment, we performed conventional molecular dynamics simulations of the apo-enzyme at two different temperatures (300 and 350 K). Our simulations show that the crystallographic <i>B</i>-factors considerably underestimate the loop dynamics; multiple conformations of loops 2 and 3, including the open, semi-open, and closed conformations that an enzyme must adopt throughout its catalytic cycle, are all accessible to the apo-enzyme. These results revise our previous view of the functional mechanism of conformational change upon MgATP binding and offer valuable structural insights into the workings of HPPK. In this paper, conformational network analysis and principal component analysis related to the loops are discussed to support the presented conclusions

Catalytic Mechanism of Yeast Cytosine Deaminase: An ONIOM Computational Study

The complete path for the deamination reaction catalyzed by yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, has been investigated using the ONIOM method. Cytosine deamination proceeds via a sequential mechanism involving the protonation of N3, the nucleophilic attack of C4 by the zinc-coordinated hydroxide, and the cleavage of the C4−N4 bond. The last step is the rate determining step for the generation of the zinc bound uracil. Uracil is liberated from the Zn atom by an oxygen exchange mechanism that involves the formation of a gem-diol intermediate from the Zn bound uracil and a water molecule, the C4−OZn cleavage, and the regeneration of the Zn-coordinated water. The rate determining step in the oxygen exchange is the formation of the gem-diol intermediate, which is also the rate determining step for the overall yCD-catalyzed deamination reaction