Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy

Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ≅15 μs relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.     

E. coli 5'-nucleotidase undergoes a hinge-bending domain rotation resembling a ball-and-socket motion

Structures of nine independent conformers of E. coli 5'-nucleotidase (5'-NT) have been analyzed using four different crystal forms. These data show that the two-domain protein undergoes an unusual 96 degrees hinge-bending domain rotation. Structures of the open and closed forms with substrates and inhibitors reveal that the substrate moves by approximately 25 A with the large domain rotation into the catalytic site. The domain motions derived from a comparison of the nine conformations agree well with motions obtained from a normal mode analysis in that all independent domain rotations are around axes that are roughly located in the plane which includes the domain centers and the hinge. Two residues, Lys355 and Gly356, form the core of the hinge region and undergo a large change of the main-chain torsion angles. The hinge-bending movement observed for 5'-nucleotidase differs markedly from a classical hinge-bending closure motion which involves an opening of the substrate or ligand-binding cleft between two domains. In contrast, the movement observed in 5'-nucleotidase resembles that of a ball-and-socket joint. The smaller C-terminal domain rotates approximately around its center such that the residues at the domain interface move in a sliding motion along the interface. Few direct interdomain contacts and a layer of water molecules between the two domains facilitate the sliding motion.     

The essential dynamics of thermolysin: Confirmation of the hinge-bending motion and comparison of simulations in vacuum and water

Comparisons of the crystal structures of thermolysin and the thermolysin-like protease produced by B. cereus have recently led to the hypothesis that neutral proteases undergo a hinge-bending motion. We have investigated this hypothesis by analyzing molecular dynamics simulations of thermolysin in vacuum and water, using the essential dynamics method. This method is able to extract large concerted atomic motions of biological importance from a molecular dynamics trajectory. The analysis of the thermolysin trajectories indeed revealed a large rigid body hinge-bending motion of the Nterminal and C-terminal domains, similar to the motion hypothesized from the crystal structure comparisons. In addition, it appeared that the essential dynamics properties derived from the vacuum simulation were similar to those derived from the solvent simulation. © 1995 Wiley-Liss, Inc.     

The blind search for the closed states of hinge-bending proteins

The hinge-bending proteins provide the most pronounced example of the large-amplitude slow motions in a number of proteins, which are critical for their functioning. They are often used as the test ground for developing novel approaches to the simulation of slow protein dynamics. In the present study, we present the algorithm, which allows physically-consistent simulations of slow protein dynamics in globular proteins. Our algorithm is based on the hierarchical clustering of the correlation patterns (HCCP) technique of domain identification, which allows subdividing the protein into the hierarchy of the rigid-body-like clusters. The clusters are allowed to rotate relative to one another on the automatically identified hinges. The clusters are found in the course of automated, objective and well-tested procedure. In the present communication, our technique is applied to 10 hinge-bending proteins. For each of the proteins, we performed the blind search for the closed conformation, staring from the open one. Resulting closed conformations are compared with the closed states observed in crystallographic structures. It is shown that our technique produces realistic closed conformations for 8 out of 10 studied proteins. This demonstrates that HCCP technique can be used for finding alternative protein conformations and for sampling the slow motions in proteins.     

Normal mode analysis of mouse epidermal growth factor: Characterization of the harmonic motion

Normal mode analysis of mouse epidermal growth factor (mEGF) has been carried out at room temperature. The value of the lowest frequency is 4.1 cm^?1. This mode corresponds to hinge-bending motion between the N-terminal and C-terminal domains of mEGF. This hinge-bending motion corresponds to the “mitten mode.” In this motion, the N-terminal domain is almost rigid. However, the C-terminal domain is found to consist of three rigid segments. Two segments, C33-D46 and G51-R53, are observed moving in the same direction, but L47-W50 moves in the opposite direction. For this mode, the effective Young's modulus turned out to be 1.1 × 10⁹ dyn·^?2. This value is a little larger than that of the mode with the lowest frequency 4.4 cm^?1 of BPTI. The difference may be related to the fraction of residues involved in β-sheets in the molecule. Similar analyses are carried out for other low frequency modes.     

Investigation of the mechanism of domain closure in citrate synthase by molecular dynamics simulation

Six, 2 ns molecular dynamics simulations have been performed on the homodimeric enzyme citrate synthase. In three, both monomers were started from the open, unliganded X-ray conformation. In the remaining three, both monomers started from a closed, liganded X-ray conformation, with the ligands removed. Projecting the motion from the simulations onto the experimental domain motion revealed that the free-energy profile is rather flat around the open conformation, with steep sides. The most closed conformations correspond to hinge-bending angles of 12-14 compared to the 20 degrees that occurs upon the binding of oxaloacetate. It is also found that the open, unliganded X-ray conformation is situated at the edge of the steep rise in free energy, although conformations that are about 5 degrees more open were sampled. A rigid-body essential dynamics analysis of the combined open trajectories has shown that domain motions in the direction of the closed X-ray conformation are compatible with the natural domain motion of the unliganded protein, which has just two main degrees of freedom. The simulations starting from the closed conformation suggest a free-energy profile with a small barrier in going from the closed to open conformation. A combined essential dynamics and hinge-bending analysis of a trajectory that spontaneously converts from the closed to open state shows an almost exact correspondence to the experimental transition that occurs upon ligand binding. The simulations support the conclusion from an earlier analysis of the experimental transition that the beta-hairpin acts as a mechanical hinge by attaching the small domain to the large domain through a conserved main-chain hydrogen bond and salt-bridges, and allowing rotation to occur via its two flexible termini. The results point to a mechanism of domain closure in citrate synthase that has analogy to the process of closing a door.     

Molecular dynamics of hinge-bending motion of IgG vanishing with hydrolysis by papain

We have performed dielectric relaxation measurements via a time domain reflectometry (TDR) method to study dynamic behaviors of the segmental flexibility of immunoglobulin G (IgG) in aqueous solution without antigen binding. In general, an intermediate relaxation process due to bound water is observed around 100 MHz at 25 degrees C for common proteins between two relaxation processes due to overall rotation and reorientation of free water. However, the intermediate process observed around 6 MHz for IgG was due to both bound water and hinge-bending motion. The apparent activation energy of 33 kJ/mol was larger than 27 kJ/mol for only bound water, and the relaxation strength was about five times as large as expected for bound water. The shape of the relaxation curve was very broad and asymmetric. These characteristic differences arising from the hinge-bending motion of IgG disappeared for fragments decomposed from IgG hydrolyzed by papain, since the hinge-bending motion did not exist in this case. We have separated the relaxation processes due to hinge-bending motion and bound water for IgG and obtained the Fab-Fab angle of IgG as about 130 degrees by Kirkwood's correlation parameter and the activation energy of 34 kJ/mol for hinge-bending motion.     

Hinge-bending deformation of enzyme observed by microwave dielectric measurement

A dielectric relaxation peak due to an intramolecular motion in the active site of trypsin was first observed in aqueous solution below the freezing temperature of bulk water by a time domain reflectometry method. If trypsin inhibitor is added to the solution, it vanishes. It is suggested that the motion observed is a hinge-bending deformation giving rise to the enzymatic activity of trypsin, which is prohibited by linkage of the trypsin inhibitor. Relaxation time of the motion is 3 × 10² ns at − 10°C. © 1996 John Wiley & Sons, Inc.     

The hinge-bending mode of a lysozyme-inhibitor complex

The hinge-bending mode of hen egg white lysozyme is studied by a constrained minimization technique. Results with and without a bound inhibitor, tri-N-acetyl-glucosamine, are obtained. The frequency of the mode with the inhibitor is found to be 4.3 cm^?1, in contrast to 3.0 cm^?1 for the free enzyme. Also, the hinge-bending angle with the lowest energy is shifted 10° towards a more closed cleft in the bound species. The main contribution to these differences arise from interactions with the residues lining the cleft and those on the back side of it. Structural details that account for the energetics are presented. The method of calculation is somewhat different from a previous study [J. A. McCammon, B. R. Gelin, M. Karplus & P. G. Wolynes, (1976) Nature 262 , 325–326] to reduce the likelihood of artifacts in the results.     

Folding funnels and conformational transitions via hinge-bending motions

In this article we focus on presenting a broad range of examples illustrating low-energy transitions via hinge-bending motions. The examples are divided according to the type of hinge-bending involved; namely, motions involving fragments of the protein chains, hinge-bending motions involving protein domains, and hinge-bending motions between the covalently unconnected subunits. We further make a distinction between allosterically and nonallosterically regulated proteins. These transitions are discussed within the general framework of folding and binding funnels. We propose that the conformers manifesting such swiveling motions are not the outcome of “induced fit” binding mechanism; instead, molecules exist in an ensemble of conformations that are in equilibrium in solution. These ensembles, which populate the bottoms of the funnels,a priori contain both the “open” and the “closed” conformational isomers. Furthermore, we argue that there are no fundamental differences among the physical principles behind the folding and binding funnels. Hence, there is no basic difference between funnels depicting ensembles of conformers of single molecules with fragment, or domain motions, as compared to subunits in multimeric quaternary structures, also showing such conformational transitions. The difference relates only to the size and complexity of the system. The larger the system, the more complex its corresponding fused funnel(s). In particular, funnels associated with allosterically regulated proteins are expected to be more complicated, because allostery is frequently involved with movements between subunits, and consequently is often observed in multichain and multimolecular complexes. This review centers on the critical role played by flexibility and conformational fluctuations in enzyme activity. Internal motions that extend over different time scales and with different amplitudes are known to be essential for the catalytic cycle. The conformational change observed in enzyme-substrate complexes as compared to the unbound enzyme state, and in particular the hinge-bending motions observed in enzymes with two domains, have a substantial effect on the enzymatic catalytic activity. The examples we review span the lipolytic enzymes that are particularly interesting, owing to their activation at the water-oil interface; an allosterically controlled dehydrogenase (lactate dehydrogenase); a DNA methyltransferase, with a covalently-bound intermediate; large-scale flexible loop motions in a glycolytic enzyme (TIM); domain motion in PGK, an enzyme which is essential in most cells, both for ATP generation in aerobes and for fermentation in anaerobes; adenylate kinase, showing large conformational changes, owing to their need to shield their catalytic centers from water; a calcium-binding protein (calmodulin), involved in a wide range of cellular calcium-dependent signaling; diphtheria toxin, whose large domain motion has been shown to yield “domain swapping” the hexameric glutamate dehydrogenase, which has been studied both in a thermophile and in a mesophile; an allosteric enzyme, showing subunit motion between the R and the T states (aspartate transcarbamoylase), and the historically well-studied lac represoor. Nonallosteric subunit transitions are also addressed with some examples (aspartate receptor andBamHI endonuclease). Hence, using this enzyme-catalysis-centered discussion, we address energy funnel landscapes of large-scale conformational transitions, rather than the faster, quasi-harmonic, thermal fluctuations.     

Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs.     

Molecular mechanism of the allosteric enhancement of the umami taste sensation

The fifth taste quality, umami, arises from binding of glutamate to the umami receptor T1R1/T1R3. The umami taste is enhanced several-fold upon addition of free nucleotides such as guanosine-5'-monophosphate (GMP) to glutamate-containing food. GMP may operate via binding to the ligand-binding domain of the T1R1 part of the umami receptor at an allosteric site. Using molecular dynamics simulations, we show that GMP can stabilize the closed (active) state of T1R1 by binding to the outer vestibule of the so-called Venus flytrap domain of the receptor. The transition between the closed and open conformations was accessed in the simulations. Using principal component analysis, we show that the dynamics of the Venus flytrap domain along the hinge-bending motion that activates signaling is dampened significantly upon binding of glutamate, and further slows down upon binding of GMP at an allosteric site, thus suggesting a molecular mechanism of cooperativity between GMP and glutamate.     

Release of ADP from the catalytic subunit of protein kinase A: a molecular dynamics simulation study

Substrate phosphorylation by cAMP-dependent-protein kinase A (protein kinase A, PKA) has been studied extensively. Phosphoryl transfer was found to be fast, whereas ADP release was found to be the slow, rate-limiting step. There is also evidence that ADP release may be preceded by a partially rate-limiting conformational change. However, the atomic details of the conformational change and the mode of ADP release are difficult to obtain experimentally. In this work, we studied ADP release from PKA by carrying out molecular dynamics simulations with different pulling forces applied to the ligand. The detailed ADP release pathway and the associated conformational changes were analyzed. The ADP release process was found to involve a swinging motion with the phosphate of ADP anchored to the Gly-rich loop, so that the more buried adenine base and ribose ring came out before the phosphate. In contrast to the common belief that a hinge-bending motion was responsible for the opening of the ligand-binding cleft, our simulations showed that the small lobe exhibited a large amplitude "rocking" motion when the ligand came out. The largest conformational change of the protein was observed at about the first quarter time point along the release pathway. Two prominent intermediate states were observed in the release process.     

Proline-induced hinges in transmembrane helices: possible roles in ion channel gating

A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.     

The influence of interdomain interactions on the intradomain motions in yeast phosphoglycerate kinase: a molecular dynamics study

A 3-ns molecular dynamics simulation in explicit solvent was performed to examine the inter- and intradomain motions of the two-domain enzyme yeast phosphoglycerate kinase without the presence of substrates. To elucidate contributions from individual domains, simulations were carried out on the complete enzyme as well as on each isolated domain. The enzyme is known to undergo a hinge-bending type of motion as it cycles from an open to a closed conformation to allow the phosphoryl transfer occur. Analysis of the correlation of atomic movements during the simulations confirms hinge bending in the nanosecond timescale: the two domains of the complete enzyme exhibit rigid body motions anticorrelated with respect to each other. The correlation of the intradomain motions of both domains converges, yielding a distinct correlation map in the enzyme. In the isolated domain simulations—in which interdomain interactions cannot occur—the correlation of domain motions no longer converges and shows a very small correlation during the same simulation time. This result points to the importance of interdomain contacts in the overall dynamics of the protein. The secondary structure elements responsible for interdomain contacts are also discussed.     

Structural insights into the autoinhibition and posttranslational activation of histone methyltransferase SmyD3

The SmyD family represents a new class of chromatin regulators that is important in heart and skeletal muscle development. However, the critical questions regarding how they are regulated posttranslationally remain largely unknown. We previously suggested that the histone methyltransferase activity of SmyD1, a vital myogenic regulator, appears to be regulated by autoinhibition and that the possible hinge motion of the conserved C-terminal domain (CTD) might be central to the maintenance and release of the autoinhibition. However, the lack of direct evidence of the hinge motion has limited our further understanding of this autoinhibitory mechanism. Here, we report the crystal structure of full-length SmyD3 in complex with the methyltransferase inhibitor sinefungin at 1.7 Å. SmyD3 has a two-lobed structure with the substrate binding cleft located at the bottom of a  15-Å-deep crevice formed between the N- and C-terminal lobes. Comparison of SmyD3 and SmyD1 clearly suggests that the CTD can undergo a large hinge-bending motion that defines two distinct conformations: SmyD3 adopts a closed conformation with the CTD partially blocking the substrate binding cleft; in contrast, SmyD1 appears to represent an open form, where the CTD swings out by ∼ 12 Å from the N-terminal lobe, forming an open cleft with the active site completely exposed. Overall, these findings provide novel structural insights into the mechanism that modulates the activity of the SmyD proteins and support the observation that a posttranslational activation, such as by molecular chaperon Hsp90, is required to potentiate the proteins.     

Rôle du changement de conformation du CD4 lors de la fusion VIH/cellule

It is well known that the gp120-gp41 complex undergoes a conformational change after CD4 binding. It is likely that CD4 undergoes a conformational change as well. Recently, a calculation of the normal modes of the two N-terminal domains of CD4 has shown that a hinge-bending motion of one of these domains with respect to the other may occur. In the present study, results obtained previously are verified with two other normal mode calculations, starting from crystallographic structures of different origin. A scheme describing the first steps of the process leading to cell infection by human immunodeficiency virus (HIV) is then proposed. It rests upon the idea that CD4 and gp120-gp41 conformational changes allow for bringing the cell and virus membranes closer to each other.