Ornithine-binding Protein Research Articles

Integrating solvation shell structure in experimentally driven molecular dynamics using x-ray solution scattering data

In the past few decades, prediction of macromolecular structures beyond the native conformation has been aided by the development of molecular dynamics (MD) protocols aimed at exploration of the energetic landscape of proteins. Yet, the computed structures do not always agree with experimental observables, calling for further development of the MD strategies to bring the computations and experiments closer together. Here, we report a scalable, efficient MD simulation approach that incorporates an x-ray solution scattering signal as a driving force for the conformational search of stable structural configurations outside of the native basin. We further demonstrate the importance of inclusion of the hydration layer effect for a precise description of the processes involving large changes in the solvent exposed area, such as unfolding. Utilization of the graphics processing unit allows for an efficient all-atom calculation of scattering patterns on-the-fly, even for large biomolecules, resulting in a speed-up of the calculation of the associated driving force. The utility of the methodology is demonstrated on two model protein systems, the structural transition of lysine-, arginine-, ornithine-binding protein and the folding of deca-alanine. We discuss how the present approach will aid in the interpretation of dynamical scattering experiments on protein folding and association.

Computational Study on Conformational Relaxation Dynamics of a Protein

Recent advances in various experimental and computational techniques have revealed atomic details of protein conformational changes and their molecular mechanisms. One of interesting features is that pathways of a large conformational change of a protein are not always identical but rather show variability because of the protein's intrinsic flexibility. However, the underlying molecular details remain unclear. In the present study, we characterized conformational relaxation dynamics of a protein using molecular dynamics (MD) simulations. As a target protein, we selected lysine-, arginine-, ornithine-binding protein (LAO), which comprises two domains and undergoes slow and large-amplitude domain motions. MD simulations of apo-LAO from the closed conformation in explicit water were repeated many times starting from the same initial coordinates but with different initial random velocities using MARBLE and the CHARMM22/CMAP force field parameters. Almost all trajectories showed conformational relaxation from the closed form to the open form, and their relaxation times were obviously different; some relaxations occur immediately, others slowly. We will analyze a variety of pathways of conformational changes using the time-structure based independent component analysis (tICA) and a Markov state model (MSM).

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis.

Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.

Slow dynamics of a protein backbone in molecular dynamics simulation revealed by time-structure based independent component analysis

We recently proposed the method of time-structure based independent component analysis (tICA) to examine the slow dynamics involved in conformational fluctuations of a protein as estimated by molecular dynamics (MD) simulation [Y. Naritomi and S. Fuchigami, J. Chem. Phys. 134, 065101 (2011)]. Our previous study focused on domain motions of the protein and examined its dynamics by using rigid-body domain analysis and tICA. However, the protein changes its conformation not only through domain motions but also by various types of motions involving its backbone and side chains. Some of these motions might occur on a slow time scale: we hypothesize that if so, we could effectively detect and characterize them using tICA. In the present study, we investigated slow dynamics of the protein backbone using MD simulation and tICA. The selected target protein was lysine-, arginine-, ornithine-binding protein (LAO), which comprises two domains and undergoes large domain motions. MD simulation of LAO in explicit water was performed for 1 μs, and the obtained trajectory of C(α) atoms in the backbone was analyzed by tICA. This analysis successfully provided us with slow modes for LAO that represented either domain motions or local movements of the backbone. Further analysis elucidated the atomic details of the suggested local motions and confirmed that these motions truly occurred on the expected slow time scale.

Rare Events in Protein Simulation Revealed by using Time-Structure Based Independent Component Analysis

Molecular dynamics (MD) simulation is a powerful tool and widely used to elucidate dynamic behavior of proteins. Protein motions occur over a wide range of time scales, but not all are important for protein functions, which time scales are generally longer. Thus, it would be reasonable to consider that slower motions of proteins are more relevant to their functions. To identify such slow protein dynamics from simulation results, we have proposed the time-structure based independent component analysis (tICA) and have validated its usefulness. It was found that the motions detected by tICA were classified into two types: slowly changing motions and rare events. In the present study, we focused on rare events in protein simulation and investigated their variety and robustness. As a target protein, we selected lysine-, arginine-, ornithine-binding protein (LAO), which undergoes a large structural change upon ligand binding. One-microsecond MD simulation of apo-LAO in explicit water was performed three times using MARBLE and the CHARMM22/CMAP force field parameters. By applying the tICA to the simulation results, several rare events were identified as local motions, and confirmed with additional analyses to be actually occurred. We will discuss their underlying mechanism and functional significance.

Slow Dynamics in Protein Fluctuations Revealed by Time-Structure Based Independent Component Analysis

Protein fluctuations in equilibrium are an important factor for its structural change and function. It has been found that a small number of low-frequency modes in a protein, determined by normal model analysis or principal component analysis, account for its large fluctuations and contribute to its structural change significantly. However, dynamical aspects of protein fluctuations and their roles in protein function remain unclear. Recently, we have proposed the time-structure based independent component analysis (tICA) to reveal slow dynamics of a protein [Y. Naritomi and S. Fuchigami, J. Chem. Phys. 134, 065101 (2011)]. In this paper, we focused on domain motions of the target protein by using rigid-body domain analysis. However, a protein shows not only domain motions but also a variety of motions of main- and side-chains. In the present study, we investigated protein main-chain dynamics on a long time scale by using all-atom molecular dynamics (MD) simulation and the tICA. As a target protein, we selected lysine-, arginine-, ornithine-binding protein (LAO), which undergoes a large structural change upon ligand binding. A one-microsecond MD simulation of apo-LAO in explicit water was performed using MARBLE and the CHARMM22/CMAP force field parameters. Applying the tICA to the simulation result yielded slow modes of the LAO, which represented both domain and local motions. Other analyses confirmed that these motions were actually occurred on the expected slow time scale, suggesting that the tICA is powerful for analyzing slow dynamics of proteins.

A role for both conformational selection and induced fit in ligand binding by the LAO protein.

Molecular recognition is determined by the structure and dynamics of both a protein and its ligand, but it is difficult to directly assess the role of each of these players. In this study, we use Markov State Models (MSMs) built from atomistic simulations to elucidate the mechanism by which the Lysine-, Arginine-, Ornithine-binding (LAO) protein binds to its ligand. We show that our model can predict the bound state, binding free energy, and association rate with reasonable accuracy and then use the model to dissect the binding mechanism. In the past, this binding event has often been assumed to occur via an induced fit mechanism because the protein's binding site is completely closed in the bound state, making it impossible for the ligand to enter the binding site after the protein has adopted the closed conformation. More complex mechanisms have also been hypothesized, but these have remained controversial. Here, we are able to directly observe roles for both the conformational selection and induced fit mechanisms in LAO binding. First, the LAO protein tends to form a partially closed encounter complex via conformational selection (that is, the apo protein can sample this state), though the induced fit mechanism can also play a role here. Then, interactions with the ligand can induce a transition to the bound state. Based on these results, we propose that MSMs built from atomistic simulations may be a powerful way of dissecting ligand-binding mechanisms and may eventually facilitate a deeper understanding of allostery as well as the prediction of new protein-ligand interactions, an important step in drug discovery.

Slow dynamics in protein fluctuations revealed by time-structure based independent component analysis: The case of domain motions

Protein dynamics on a long time scale was investigated using all-atom molecular dynamics (MD) simulation and time-structure based independent component analysis (tICA). We selected the lysine-, arginine-, ornithine-binding protein (LAO) as a target protein and focused on its domain motions in the open state. A MD simulation of the LAO in explicit water was performed for 600 ns, in which slow and large-amplitude domain motions of the LAO were observed. After extracting domain motions by rigid-body domain analysis, the tICA was applied to the obtained rigid-body trajectory, yielding slow modes of the LAO's domain motions in order of decreasing time scale. The slowest mode detected by the tICA represented not a closure motion described by a largest-amplitude mode determined by the principal component analysis but a twist motion with a time scale of tens of nanoseconds. The slow dynamics of the LAO were well described by only the slowest mode and were characterized by transitions between two basins. The results show that tICA is promising for describing and analyzing slow dynamics of proteins.

Molecular dynamics simulations of ligand-induced backbone conformational changes in the binding site of the periplasmic lysine-, arginine-, ornithine-binding protein

The periplasmic lysine-, arginine-, ornithine-binding protein (LAOBP) traps its ligands by a large hinge bending movement between two globular domains. The overall geometry of the binding site remains largely unchanged between the open (unliganded) and closed (liganded) forms, with only a small number of residues exhibiting limited movement of their side chains. However, in the case of the ornithine-bound structure, the backbone peptide bond between Asp11 and Thr12 undergoes a large rotation. Molecular dynamics simulations have been used to investigate the origin and mechanism of this backbone movement. Simulations allowing flexibility of a limited region and of the whole binding site, with and without bound ligands, suggest that this conformational change is induced by the binding of ornithine, leading to the stabilisation of an energetically favourable alternative conformation.

A probe molecule composed of seventeen percent of total diffracting matter gives correct solutions in molecular replacement.

It is often found in the crystallization of enzyme-inhibitor complexes that an inhibitor causes crystal packing which is different to that of native protein. This is the case for crystals of human non-pancreatic secreted phospholipase A(2) (124 residues) containing six molecules in the asymmetric unit when the protein is complexed with a potential acylamino analogue of a phospholid. The hexameric structure was determined by molecular replacement using the structure of monomeric native protein as a probe. As an extension to the experiment, it was tested whether a backbone polypeptide composed of 17% of a known monomeric structure could find its correct position on a target molecule in molecular replacement. A probe model composed of the backbone atoms of the N-terminal 77 residues of lysine-, arginine-, ornithine-binding protein (LAO, a total of 238 residues) liganded with lysine correctly finds its position on LAO liganded with histidine which crystallizes as a monomer in the asymmetric unit. The results indicate that as little as 17% of total diffracting matter can be used in molecular replacement to solve crystal structures or to obtain phase information which can be combined with phases obtained by the isomorphous-replacement method.

Structural basis for multiple ligand specificity of the periplasmic lysine-, arginine-, ornithine-binding protein

The substrate-binding site of a protein with multiple specificity should satisfy geometric and energetic complementarity for several different substrates. The structural basis of the multiple ligand specificity of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) was investigated by determining and analyzing the structures of the protein unliganded and liganded with each of the three high-affinity ligands (L-lysine, L-arginine, and L-ornithine) and with one low-affinity ligand (L-histidine). The geometric complementarity is achieved primarily by virtue of the large size of the ligand-binding site which can accommodate the maximum common volume of the four ligands plus three water molecules. The optimization of energetic complementarity is achieved by the relocation of protein-bound water molecules and by the movement of the Asp-11 side chain. The structure of the LAO-histidine complex indicates that the 30-fold reduced affinity of the protein for histidine is primarily due to unavailability of one ionic interaction of the histidine side chain with the protein which is present in the other three complexes.

The histidine-binding protein undergoes conformational changes in the absence of ligand as analyzed with conformation-specific monoclonal antibodies.

The periplasmic histidine-binding protein, HisJ, and the lysine-, arginine-, ornithine-binding protein (LAO) are receptors for histidine transport via the histidine permease of Salmonella typhimurium. The receptors have similar structures, being composed of two lobes held together by two peptide segments, with the ligand-binding site located in a cleft between the lobes. The two lobes are far apart in the unliganded structure (open conformation) and are drawn close together in the liganded structure (closed conformation). The tight binding of the ligand via protein side chains as well as the peptide backbone from both lobes stabilizes the closed conformation (Oh, B.-H., Pandit, J., Kang, C.-H., Nikaido, K., Gokcen, S., Ames, G. F.-L., and Kim, S.-H. (1993) J. Biol. Chem. 268, 11348-11353; Oh, B.-H., Kang, C.-H., De Bondt, H., Kim, S.-H., Nikaido, K., Joshi, A., and Ames, G. F.-L. (1994) J. Biol. Chem. 269, 4135-4143). In this study two conformation-specific monoclonal antibodies (mAbs) that trap the protein in the closed empty form have been characterized and used to provide evidence that HisJ can assume the closed empty form in the absence of ligand. Several pieces of evidence were provided to demonstrate that these mAbs are specific for HisJ in the closed form. Histidine improves the interaction of these mAbs with immobilized HisJ. The mAbs inhibit both the exchange and the dissociation of histidine from HisJ, indicating that they are able to trap the protein in the closed liganded form. The characterization of the epitopes of the conformation-specific mAbs shows that they include residues that are located in both lobes and that are far apart in the open form but close to each other in the closed form, so that the mAbs must be sensitive to their spatial orientation. Two mAbs that are not conformation-specific according to these criteria were also identified.

The bacterial periplasmic histidine-binding protein. structure/function analysis of the ligand-binding site and comparison with related proteins.

Bacterial periplasmic binding proteins are initial receptors in the process of active transport across cell membranes and/or chemotaxis. Among them, the histidine-binding protein (HisJ) has been extensively studied from the biochemical, physiological, and genetic points of view. The three-dimensional crystal structure of the histidine-binding protein complexed with histidine has been determined at 2.5-A resolution by the molecular replacement method using a probe structure the previously solved lysine-liganded structure of the lysine-, arginine-, ornithine-binding protein (LAO), which shares 70% sequence identity with HisJ. The structure is bi-lobate; the two lobes, one bigger than the other, are connected by two short strands and are in contact with each other (closed) enclosing the histidine. Charged, polar, and non-polar side chains, as well as the peptide backbone, are involved in tight binding of the histidine. The bound histidine is involved in eight direct hydrogen bonds, six with the bigger lobe and two with the smaller lobe, in one potential water-mediated hydrogen bond with the bigger lobe, as well as in ionic interactions. The HisJ residues surrounding the ligand are the same as the LAO residues interacting with lysine, except for residue 52 which is leucine in HisJ and phenylalanine in LAO. The Leu-52 in HisJ makes a hydrophobic interaction with the imidazole ring of histidine. Of seven mutations affecting the ligand-binding site, five are located in the ligand-binding site, one in a connecting strand, and one at the domains interface. Based on comparisons among related binding proteins, the specific interactions between the ligands and the respective binding protein residues are predicted for the glutamine-binding protein and the opines-binding protein.

Purification and characterization of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium.

The lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium has been purified to homogeneity and characterized. The dissociation constants (KD) were determined by equilibrium dialysis assay to be 14, 15, and 29 nM for L-arginine, L-lysine, and L-ornithine respectively. L-Histidine was found to be a relatively good ligand (KD, 500 nM). Methods have been developed for the separation of liganded from unliganded LAO, for the estimation of bound ligand, and for unliganding LAO. Liganded and unliganded LAO are shown to have distinct UV spectra. The UV spectrum also varies with the nature of the substrate. Inhibition studies with substrate analogs yielded information useful for understanding the nature of the ligand-binding pocket.

Crystallization and preliminary X-ray studies of the liganded lysine, arginine, ornithine-binding protein from Salmonella typhimurium

The periplasmic binding protein LAO from Salmonella typhimurium, which is involved in lysine, arginine and ornithine transport, has been crystallized together with one of its ligands, arginine (LAO-Arg). Preliminary X-ray diffraction studies of LAO-Arg crystal show that it belongs to the orthorhombic space group P2 12 12 1 and has the unit cell dimensions of a = 37.65 A ̊ , b = 59.45 A ̊ , c = 115.91 A ̊ . Crystals of the LAO-Arg complex diffract beyond 2.0 Å resolution.

Crystal structure of the lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium at 2.7-A resolution.

A wide variety of sugars, amino acids, peptides, and inorganic ions are transported into bacteria by periplasmic transport systems consisting of substrate-specific receptors (binding proteins) and membrane-bound protein complexes. The crystal structure of the lysine-, arginine-, ornithine-binding protein (LAO) at 2.7-A resolution shows that the molecule has a bi-lobal structure and that its topological structure is different from other amino acid-binding proteins but is similar to the sulfate-binding protein and maltose-binding protein. High sequence homology between LAO and the histidine-binding protein (HisJ) and the fact that LAO and HisJ share the same membrane-bound protein complex allow one to define functional regions responsible for the ligand binding and for the interaction with the membrane complex.

Salmonella typhimurium histidine periplasmic permease mutations that allow transport in the absence of histidine-binding proteins

Periplasmic transport systems consist of a membrane-bound complex and a periplasmic substrate-binding protein and are postulated to function by translocating the substrate either through a nonspecific pore or through specific binding sites located in the membrane complex. We have isolated mutants carrying mutations in one of the membrane-bound components of the histidine permease of Salmonella typhimurium that allow transport in the absence of both histidine-binding proteins HisJ and LAO (lysine-, arginine-, ornithine-binding protein). All of the mutations are located in a limited region of the nucleotide-binding component of the histidine permease, HisP. The mutants transported substrate in the absence of binding proteins only when the membrane-bound complex was produced in large amounts. At low (chromosomal) levels, the mutant complex was unable to transport substrate in the absence of binding proteins but transported it efficiently in the presence of HisJ. The alterations responsible for the mutations were identified by DNA sequencing; they are closely related to a group of hisP mutations isolated as suppressors of HisJ interaction mutations (G. F.-L. Ames and E. N. Spudich, Proc. Natl. Acad. Sci. USA 73:1877-1881, 1976). The hisP suppressor mutations behaved similarly to these newly isolated mutations despite the entirely different selection procedure. The results are consistent with the HisP protein carrying or contributing to the existence of a substrate-binding site that can be mutated to function in the absence of a binding protein.