Induced-fit Motion Research Articles

Relationship between the induced-fit loop and the activity of Klebsiella pneumoniae pullulanase.

Klebsiella pneumoniae pullulanase (KPP) belongs to glycoside hydrolase family 13 subfamily 13 (GH13_13) and is the only enzyme that is reported to perform an induced-fit motion of the active-site loop (residues 706-710). Comparison of pullulanase structures indicated that only KPP has Leu680 present behind the loop, in contrast to the glycine found in other GH13_13 members. Analysis of the structure and activity of recombinant pullulanase from K. pneumoniae ATCC 9621 (rKPP) and its mutant (rKPP-G680L) indicated that the side chain of residue 680 is important for the induced-fit motion of the loop 706-710 and alters the binding affinity of the substrate.

Heterogeneous and rate-dependent streptavidin-biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations.

Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information on the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin-biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket, while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and rate-dependent induced-fit motions for intermediates, which might be relevant for other receptor-ligand bonds.

High-Pressure NMR Spectroscopy Reveals Functional Sub-states of Ubiquitin and Ubiquitin-Like Proteins.

High-pressure nuclear magnetic resonance (NMR) spectroscopy has revealed that ubiquitin has at least two high-energy states--an alternatively folded state N2 and a locally disordered state I--between the basic folded state N1 and totally unfolded U state. The high-energy states are conserved among ubiquitin-like post-translational modifiers, ubiquitin, NEDD8, and SUMO-2, showing the E1-E2-E3 cascade reaction. It is quite intriguing that structurally similar high-energy states are evolutionally conserved in the ubiquitin-like modifiers, and the thermodynamic stabilities vary among the proteins. To investigate atomic details of the high-energy states, a Q41N mutant of ubiquitin was created as a structural model of N2, which is 71% populated even at atmospheric pressure. The convergent structure of the "pure" N2 state was obtained by nuclear Overhauser effect (NOE)-based structural analysis of the Q41N mutant at 2.5 kbar, where the N2 state is 97% populated. The N2 state of ubiquitin is closely similar to the conformation of the protein bound to the ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is best explained by conformational selection rather than by induced-fit motion.

On the induced-fit mechanism of substrate-enzyme binding structures of nylon-oligomer hydrolase.

We present a detailed computational investigation of the induced-fit motion in a nylon-oligomer hydrolase (NylB) upon substrate binding. To this aim, we resort on the recently introduced parallel cascade selection molecular dynamics approach, allowing for an accelerated access to the set of conformational changes from an open- to a closed-state structure to form the enzyme-substrate complex in a specific induce-fit mechanism. The structural investigation is quantitatively complemented by free energy analyses within the umbrella sampling algorithm accompanied by weighted histogram analysis. We find that the stabilization free energy is about 1.4 kcal/mol, whereas the highest free energy barrier to be overcome is about 2.3 kcal/mol. Conversely, the energetic contribution for the substrate binding is about 20 kcal/mol, as estimated from Generalized Born/Surface Area. This means that the open-close induced-fit motion could occur frequently once the substrate binds to the open state of NylB.

Close Identity between Alternatively Folded State N2 of Ubiquitin and the Conformation of the Protein Bound to the Ubiquitin-Activating Enzyme

We present the nuclear Overhauser effect-based structure determination of the Q41N variant of ubiquitin at 2500 bar, where the alternatively folded N2 state is 97% populated. This allows us to characterize the structure of the "pure" N2 state of ubiquitin. The N2 state shows a substantial change in the orientation of strand β5 compared to that of the normal folded N1 state, which matches the changes seen upon binding of ubiquitin to ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is therefore best explained by conformational selection rather than induced-fit motion.

Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies

BackgroundPQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ.ResultsEnzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and β-ketodecanoic acid.ConclusionsEnzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for β-ketodecanoic acid were identified with distinct catalytic mechanism preferences.

Induced-fit motion of a lid loop involved in catalysis in alginate lyase A1-III

The structures of two mutants (H192A and Y246F) of a mannuronate-specific alginate lyase, A1-III, from Sphingomonas species A1 complexed with a tetrasaccharide substrate [4-deoxy-L-erythro-hex-4-ene-pyranosyluronate-(mannuronate)(2)-mannuronic acid] were determined by X-ray crystallography at around 2.2 Å resolution together with the apo form of the H192A mutant. The final models of the complex forms, which comprised two monomers (of 353 amino-acid residues each), 268-287 water molecules and two tetrasaccharide substrates, had R factors of around 0.17. A large conformational change occurred in the position of the lid loop (residues 64-85) in holo H192A and Y246F compared with that in apo H192A. The lid loop migrated about 14 Å from an open form to a closed form to interact with the bound tetrasaccharide and a catalytic residue. The tetrasaccharide was bound in the active cleft at subsites -3 to +1 as a substrate form in which the glycosidic linkage to be cleaved existed between subsites -1 and +1. In particular, the O(η) atom of Tyr68 in the closed lid loop forms a hydrogen bond to the side chain of a presumed catalytic residue, O(η) of Tyr246, which acts both as an acid and a base catalyst in a syn mechanism.

Recognition Dynamics Up to Microseconds Revealed from an RDC-Derived Ubiquitin Ensemble in Solution

Protein dynamics are essential for protein function, and yet it has been challenging to access the underlying atomic motions in solution on nanosecond-to-microsecond time scales. We present a structural ensemble of ubiquitin, refined against residual dipolar couplings (RDCs), comprising solution dynamics up to microseconds. The ensemble covers the complete structural heterogeneity observed in 46 ubiquitin crystal structures, most of which are complexes with other proteins. Conformational selection, rather than induced-fit motion, thus suffices to explain the molecular recognition dynamics of ubiquitin. Marked correlations are seen between the flexibility of the ensemble and contacts formed in ubiquitin complexes. A large part of the solution dynamics is concentrated in one concerted mode, which accounts for most of ubiquitin's molecular recognition heterogeneity and ensures a low entropic complex formation cost.

Deduced catalytic mechanism of D-amino acid amidase from Ochrobactrum anthropi SV3.

D-Amino acid amidase (DAA) from Ochrobactrum anthropi SV3 catalyzes D-stereospecific hydrolysis of amino acid amides. DAA has attracted attention as a catalyst for the stereospecific production of D-amino acids, although the mechanism that drives the reaction has not been clear. Previously, the structure of DAA was classified into two types, a substrate-bound state with an ordered Omega loop, and a ground state with a disordered Omega loop. Because the binding of the substrate facilitates ordering, this transition was regarded to be induced fit motion. The angles and distances of hydrogen bonds at Tyr149 Oeta, Ser60 Ogamma and Lys63 Nzeta revealed that Tyr149 Oeta donates an H atom to a water molecule in the substrate-bound state, and that Tyr149 Oeta donates an H atom to Ser60 Ogamma or Lys63 Nzeta in the ground state. Taking into consideration the locations of the H atoms of Tyr149 Oeta, Ser60 Ogamma and Lys63 Nzeta, a catalytic mechanism of DAA activity is presented, wherein a shift of an H atom at Tyr149 Oeta in the substrate-bound versus the ground state plays a significant role in the reaction. This mechanism explains well why acylation proceeds and deacylation does not proceed in the substrate-bound state.

Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity.

New insights into functional flexibility at the peptidyl transferase center (PTC) and its vicinity were obtained by analysis of pleuromutilins binding modes to the ribosome. The crystal structures of Deinococcus radiodurans large ribosomal subunit complexed with each of three pleuromutilin derivatives: retapamulin (SB-275833), SB-280080, and SB-571519, show that all bind to the PTC with their core oriented similarly at the A-site and their C14 extensions pointing toward the P-site. Except for an H-bond network with a single nucleotide, G2061, which involves the essential keto group of all three compounds, only minor hydrophobic contacts are formed between the pleuromutilin C14 extensions and any ribosomal component, consistent with the PTC tolerance to amino acid diversity. Efficient drug binding mode is attained by a mechanism based on induced-fit motions exploiting the ribosomal intrinsic functional flexibility and resulting in conformational rearrangements that seal the pleuromutilin-binding pocket and tightens it up. Comparative studies identified a network of remote interactions around the PTC, indicating that pleuromutilins selectivity is acquired by nonconserved nucleotides residing in the PTC vicinity, in a fashion resembling allosterism. Likewise, pleuromutilin resistant mechanisms involve nucleotides residing in the environs of the binding pocket, consistent with their slow resistance-development rates.

Effective handling of induced‐fit motion in flexible docking

For structure-based drug design, where various ligand structures need to be docked to a target protein structure, a docking method that can handle conformational flexibility of not only the ligand, but also the protein, is indispensable. We have developed a simple and effective approach for dealing with the local induced-fit motion of the target protein, and implemented it in our docking tool, ADAM. Our approach efficiently combines the following two strategies: a vdW-offset grid in which the protein cavity is enlarged uniformly, and structure optimization allowing the motion of ligand and protein atoms. To examine the effectiveness of our approach, we performed docking validation studies, including redocking in 18 test cases and foreign-docking, in which various ligands from foreign crystal structures of complexes are docked into a target protein structure, in 22 cases (on five target proteins). With the original ADAM, the correct docking modes (RMSD < 2.0 A) were not present among the top 20 models in one case of redocking and four cases of foreign-docking. When the handling of induced-fit motion was implemented, the correct solutions were acquired in all 40 test cases. In foreign-docking on thymidine kinase, the correct docking modes were obtained as the top-ranked solutions for all 10 test ligands by our combinatorial approach, and this appears to be the best result ever reported with any docking tool. The results of docking validation have thus confirmed the effectiveness of our approach, which can provide reliable docking models even in the case of foreign-docking, where conformational change of the target protein cannot be ignored. We expect that this approach will contribute substantially to actual drug design, including virtual screening.

Conformational Changes Observed in Enzyme Crystal Structures upon Substrate Binding

The theory of induced fit predicts that enzymes undergo conformational changes as they bind their substrate. We have analysed the structures of 60 different enzymes to see if conformational changes are observed between the apo form, and the substrate (or substrate analog) bound form. In each enzyme the residues responsible for catalysis and substrate binding are known and are examined to see how the active site area is affected by conformational changes. Surprisingly, we find that induced fit motions in most enzymes is very small (usually 1 A RMSD between the apo and substrate-bound forms across the whole protein). We also find that there is a significant difference between the motions undergone by the binding residues and those undergone by the catalytic residues. The binding residues tend to exhibit larger backbone motions, but both binding and catalytic residues show the same, considerable, amount of side-chain flexibility. Knowing the extent of induced fit in enzymes is important for our understanding of the principles of enzyme catalysis and also for improving ligand docking and structural template searching.

How Does Lipase Flexibility Affect Its Enantioselectivity in Organic Solvents? A Possible Role of CH···π Association in Stabilization of Enzyme–Substrate Complex

Abstract For lipase-catalyzed reactions of 2-(4-substitued phenoxy)propionic acids with alcohols in organic solvents containing a small amount of water, the increase of the lipase flexibility brought about by addition of water is found to be favorable for the induced-fit motion of the lipase for the correctly binding enantiomer of the substrate used, thus resulting in the improvement of the lipase enantioselectivity. In particular, for the reaction of the substrate with rich π electron density on its aromatic ring, the enantioselectivity was much more sensitive to the change of the lipase flexibility. Thus, in the induced fit motion, the CH···π association between amino acids side chains around the lipase’s active site and the aromatic ring of the correctly binding enantiomer is assumed to accelerate the accommodation of the substrate into the lipase’s active site and the stabilization of the complex between the enzyme and the substrate. This assumption is also supported by a discussion based on the value of the Michaelis constant obtained. Furthermore, on the basis of a model concerning the acyl-enzyme structure for the incorrectly binding enantiomer, the long alkyl chain alcohols as a nucleophile are found to improve the lipase enantioselectivity markedly.

Stability, Activity and Structure of Adenylate Kinase Mutants

Sequence/structure relationships have been explored by site‐directed mutagenesis using a structurally known adenylate kinase. In particular the effects of helix capping and nonpolar core expansion on thermo‐dynamic stability have been analyzed. Six point mutations were produced and characterized by SDS/PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X‐ray structure analysis. Heat‐denaturation experiments yielded melting temperatures Tm and melting enthalpy changes ?Hm. The heat capacity change ?CP of the wild‐type enzyme was determined by guanidine hydrochloride denaturation in conjunction with Tm and ?Hm. Using the wild‐type ?CP value, Gibbs free energy changes ?G at room temperature were calculated for all mutants. Four mutants were designed for helix capping stabilization, but only one of them showed such an effect. Because of electrostatic interference with the induced‐fit motion, one mutant decreased the catalytic activity strongly. Two mutants expanded nonpolar cores causing destabilization. The mutant with the lower stability could be crystallized and subjected to an X‐ray analysis at 223‐pm resolution which showed the structural changes. The enzyme was stabilized by adding a ‐Pro‐His‐His tail to the C‐terminal α‐helix for nickel‐chelate chromatography. This addition constitutes a helix cap. Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption. Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing.

Stability, activity and structure of adenylate kinase mutants.

Sequence/structure relationships have been explored by site-directed mutagenesis using a structurally known adenylate kinase. In particular the effects of helix capping and nonpolar core expansion on thermodynamic stability have been analyzed. Six point mutations were produced and characterized by SDS/PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X-ray structure analysis. Heat-denaturation experiments yielded melting temperatures Tm and melting enthalpy changes delta Hm. The heat capacity change delta Cp of the wild-type enzyme was determined by guanidine hydrochloride denaturation in conjunction with Tm and delta Hm. Using the wild-type delta Cp value, Gibbs free energy changes delta G at room temperature were calculated for all mutants. Four mutants were designed for helix capping stabilization, but only one of them showed such an effect. Because of electrostatic interference with the induced-fit motion, one mutant decreased the catalytic activity strongly. Two mutants expanded nonpolar cores causing destabilization. The mutant with the lower stability could be crystallized and subjected to an X-ray analysis at 223-pm resolution which showed the structural changes. The enzyme was stabilized by adding a -Pro-His-His tail to the C-terminal alpha-helix for nickel-chelate chromatography. This addition constitutes a helix cap. Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption. Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing.

Induced-fit movements in adenylate kinases.

Adenylate kinases have an M(r) around 23,000 which classifies them among the smallest phosphoryl group transferring enzymes. In order to prevent phosphoryl transfer to water, i.e. hydrolysis, these enzymes undergo induced-fit motions on substrate binding and assemble/disassemble their catalytic centres during each reaction cycle. Details of these processes have been derived from several X-ray structure analyses. The disturbance of these analyses by crystal-packing effects is discussed.