Re-Docking Scheme to Explore Docking Search Space by using Interaction Profiles

Domain-Based Protein Docking with Extremely Large Conformational Changes

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.

Biochemistry is the science that studies the chemistry of life. This 'biological' chemistry includes growth, differentiation, movement, conductivity, immunity, transport and storage. During these processes proteins play an important role. The building blocks of proteins are amino acids, of which twenty are known. With these building blocks at hand it is possible to construct numerous proteins with many specific functions. A protein is not an elongated chain of amino acid residues but a compact very well defined three-dimensional structure. Two basic substructures are known in a protein, a cylindricalα-helix and an elongatedβ-strand. A number of theseα-helices and/orβ-strands connected by loop regions form a protein domain and a protein is built up of one or more domains. Furthermore, proteins can contain certain motifs (folds), structural conserved patterns. A large group of proteins with similar function and/or structure are called a protein family.A special group of proteins, called enzymes or biocatalysts, are able to increase the rate of a chemical reaction by lowering the activation energy of that reaction. Enzymes are highly specific, because they influence the reactivity of the substrate in such a way that the substrate is quickly and efficiently converted into a product. Moreover, flexible/dynamic movements in enzymes may play an important role during catalysis, because enzymes are not always rigid bodies. To control the reaction, enzymes often need cofactors. Some examples are the already mentioned dinucleotides NAD(P)H and FAD, that play a role in electron transfer (redox) reactions. Generally speaking, these cofactors bind very specific to a protein. A well-known binding motif for NAD(P)H and FAD in different enzyme families is the Rossmann fold (Chapter 1), discovered by Michael Rossmann in 1974.The NAD(P)H cofactor binds to the enzyme, electron transfer takes place and finally, the oxidized cofactor is released. In some proteins, the mode of NADPH binding is unknown. One example is p -hydroxybenzoate hydroxylase (PHBH), a flavoprotein monooxygenase that belongs to the family of FAD-dependent aromatic hydroxylases.FAD-dependent Aromatic HydroxylasesFAD-dependent aromatic hydroxylases play a role in the biodegradation of aromatic compounds. In nature, these compounds occur in plant polymers (lignin) as well as in proteins, steroïds and terpenes. During this century, the natural pool of aromatic compounds has been extended with products of industrial origin. Many of these synthetic compounds (pesticides, herbicides, fungicides and detergents) place a heavy burden on the environment and accumulate in soil and sludge. Microbial FAD-dependent aromatic hydroxylases catalyze the conversion of natural and synthetic aromatic substrates into products that can be further degraded to carbon dioxide and water. Recently, it was found that these enzymes are also involved in the biosynthesis of steroïds, plant hormones and antibiotics. PHBH is the archetype (prototype) of the family of FAD-dependent aromatic hydroxylases. In Wageningen, research on PHBH and related enzymes is embedded in the Wageningen Graduate School of Environmental Chemistry & Toxicology.p -Hydroxybenzoate Hydroxylasep -Hydroxybenzoate hydroxylase is isolated from the soil bacterium Pseudomonas fluorescens . This microbe can grow on 4-hydroxybenzoate (POHB) and other aromatic compounds as sole carbon source. PHBH catalyzes the conversion of POHB into 3,4-dihydroxybenzoate (DOHB) in the presence of NADPH and molecular oxygen. DOHB is a common intermediate in the aerobic degradation of plant material. After ring cleavage of DOHB and further degradation, the final products acetyl coenzyme A and succinate are fed into the citric acid cycle to provide energy for the cell.p -Hydroxybenzoate hydroxylase has been subject to detailed kinetic and structural studies. The three-dimensional structure of PHBH is built up of three domains (Chapter 1). The first domain is the FAD-binding domain with the specific Rossmann fold for binding the ADP part of FAD. The second domain is the substrate-binding domain and the third domain (interface domain) is important for the interaction with another PHBH subunit, because PHBH exists as a dimer.The structure of the enzyme-substrate complex is known in atomic detail. Recently, it was found that the flavin ring is able to move between an "open" and "closed" conformation. This flavin mobility is important for substrate binding and product release. However, unknown is the NADPH-binding site and where the reaction between NADPH and FAD takes place. Related questions are:-Which amino acids play a role in cofactor binding?-Is there a particular sequence motif for cofactor binding?Which amino acids are responsible for the coenzyme specificity and involved in binding of the 2'-phosphate moiety of NADPH?Another very important question concerns the effector role of the substrate. Upon binding of the aromatic substrate the flow of electrons from NADPH to FAD is 105 times enhanced. However, the molecular principles of this control are poorly understood. In this thesis we have tried to shed more light on the coenzyme recognition by PHBH.Flavin ring mobilityIn Chapter 2 the FAD in PHBH is substituted by a modified FAD, normally present in alcohol oxidase from methylotrophic yeasts. The crystal structure of p -hydroxybenzoate hydroxylase with this flavin analog not only represents the first crystal structure of an enzyme reconstituted with a modified flavin, but also provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The reconstituted enzyme-substrate complex shows that the flavin ring attains the "open" conformation. In the native enzyme-substrate complex the flavin ring is located in the "closed" conformation. The rate of flavin reduction by NADPH is much more rapid as compared to the native enzyme-substrate complex, suggesting that the mobility of the flavin ring is essential for the efficient reduction of the enzyme/substrate complex.Amino acids involved in NADPH bindingTo investigate the mode of NADPH binding, several amino acid residues were replaced by site-directed mutagenesis. The amino acids were selected on the basis of earlier results from chemical modification, crystallographic and modeling studies. Chapters 3, 4, 6 and 8 describe the properties of single mutants. It is concluded that Arg33, Gln34, Tyr38, Arg42, Arg44, His162 and Arg269 are involved in NADPH binding.Structural motif for NADPH bindingPHBH contains two conserved sequence motifs, both involved in FAD binding. Chapter 5 describes a new unique sequence motif for the family of FAD-dependent aromatic hydroxylases, putatively involved in both FAD and NAD(P)H binding. From the recently determined crystal structure of phenol hydroxylase it is deduced that this sequence motif is also structurally conserved. Chapter 6 and 7 show that only His162 of this novel motif is directly important for the binding of NADPH.Coenzyme specificityChapter 8 describes the cloning, purification and characterization of PHBH from Pseudomonas species CBS3. This is the first PHBH enzyme with known sequence that is active with NADH. Based on sequence analysis and homology modelling it is proposed that the helix H2 region is important for the binding of the 2'-phosphate moiety of NADPH. In Chapter 9 , the coenzyme specificity of PHBH from Pseudomonas fluorescens was addressed in further detail. Multiple replacements in helix H2 showed that Arg33 and Tyr38 are crucially involved in determining the coenzyme specificity. For the first time, a PHBH enzyme was constructed, which is more efficient with NADH.Effector specificitySubstrate binding is essential for a rapid reduction of FAD. This allows the subsequent attack of oxygen and the formation of the flavinhydroperoxide hydroxylating species. The question arises whether the stimulating effect of substrate binding on flavin reduction is caused by a large conformational change or merely due to subtle rearrangements in the active site. Chapter 10 describes the crystal structure of the substrate-free enzyme. This study shows that no large conformational changes take place upon substrate (analog) binding. The stimulating role of POHB is probably caused by several subtle effects. Stabilisation of the phenolate form of the substrate results in distribution of the electronic charges in the active site. These charge distributions influence the dynamic equilibrium between the "open" and "closed" conformation of FAD in such a way that the nicotinamide ring of NADPH and the isoalloxazine ring of FAD become optimally oriented for efficient reduction.

The important role of the serine/threonine protein phosphatase 2A (PP2A) in various cellular processes requires a precise and dynamic regulation of PP2A activity, localization, and substrate specificity. The regulation of the function of PP2A involves the reversible methylation of the COOH group of the C-terminal leucine of the catalytic subunit, which, in turn, controls the enzyme's heteromultimeric composition and confers different protein recognition and substrate specificity. We have determined the structure of PPM1, the yeast methyltransferase responsible for methylation of PP2A. The structure of PPM1 reveals a common S-adenosyl-l-methionine-dependent methyltransferase fold, with several insertions conferring the specific function and substrate recognition. The complexes with the S-adenosyl-l-methionine methyl donor and the S-adenosyl-l-homocysteine product and inhibitor unambiguously revealed the co-substrate binding site and provided a convincing hypothesis for the PP2A C-terminal peptide binding site. The structure of PPM1 in a second crystal form provides clues to the dynamic nature of the PPM1/PP2A interaction.

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12-18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.

Chapter 3 - Post-processing of Docking Results: Tools and Strategies

Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in complex with a Fab fragment of this antibody (S77) illustrates the structural basis for this selectivity. Cleavage of C3 to C3b results in a plethora of structural changes within C3, including the rearrangement of macroglobulin domain 6 enabling binding of S77 to the adjacent macroglobulin domain 7 domain. S77 blocks binding of factor B to C3b inhibiting the first step in the formation of the alternative pathway C3 convertase. In addition, S77 inhibits C5 binding to C3b. This results in significantly reduced formations of anaphylatoxins and membrane-attack complexes. This study for the first time demonstrates the structural basis for complement inhibition by a C3b-selective antibody and provides insights into the molecular mechanisms of alternative pathway complement activation.

Co-evolution has an important function in the evolution of species and it is clearly manifested in certain scenarios such as host–parasite and predator–prey interactions, symbiosis and mutualism. The extrapolation of the concepts and methodologies developed for the study of species co-evolution at the molecular level has prompted the development of a variety of computational methods able to predict protein interactions through the characteristics of co-evolution. Particularly successful have been those methods that predict interactions at the genomic level based on the detection of pairs of protein families with similar evolutionary histories (similarity of phylogenetic trees: mirrortree). Future advances in this field will require a better understanding of the molecular basis of the co-evolution of protein families. Thus, it will be important to decipher the molecular mechanisms underlying the similarity observed in phylogenetic trees of interacting proteins, distinguishing direct specific molecular interactions from other general functional constraints. In particular, it will be important to separate the effects of physical interactions within protein complexes (‘co-adaptation') from other forces that, in a less specific way, can also create general patterns of co-evolution.

It is important to extract reaction coordinates or order parameters from protein simulations in order to investigate the local minimum-energy states and the transitions between them. The most popular method to obtain such data is principal component analysis, which extracts modes of large conformational fluctuations around an average structure. We recently applied relaxation mode analysis for protein systems, which approximately estimates the slow relaxation modes and times from a simulation and enables investigations of the dynamic properties underlying the structural fluctuations of proteins. In this study, we apply this relaxation mode analysis to extract reaction coordinates for a system in which there are large conformational changes such as those commonly observed in protein folding/unfolding. We performed a 750-ns simulation of chignolin protein near its folding transition temperature and observed many transitions between the most stable, misfolded, intermediate, and unfolded states. We then applied principal component analysis and relaxation mode analysis to the system. In the relaxation mode analysis, we could automatically extract good reaction coordinates. The free-energy surfaces provide a clearer understanding of the transitions not only between local minimum-energy states but also between the folded and unfolded states, even though the simulation involved large conformational changes. Moreover, we propose a new analysis method called Markov state relaxation mode analysis. We applied the new method to states with slow relaxation, which are defined by the free-energy surface obtained in the relaxation mode analysis. Finally, the relaxation times of the states obtained with a simple Markov state model and the proposed Markov state relaxation mode analysis are compared and discussed.

Sarco(endo)plasmic reticulum Ca(2+)-ATPase transports two Ca(2+) per ATP-hydrolyzed across biological membranes against a large concentration gradient by undergoing large conformational changes. Structural studies with X-ray crystallography revealed functional roles of coupled motions between the cytoplasmic domains and the transmembrane helices in individual reaction steps. Here, we employed "Motion Tree (MT)," a tree diagram that describes a conformational change between two structures, and applied it to representative Ca(2+) -ATPase structures. MT provides information of coupled rigid-body motions of the ATPase in individual reaction steps. Fourteen rigid structural units, "common rigid domains (CRDs)" are identified from seven MTs throughout the whole enzymatic reaction cycle. CRDs likely act as not only the structural units, but also the functional units. Some of the functional importance has been newly revealed by the analysis. Stability of each CRD is examined on the morphing trajectories that cover seven conformational transitions. We confirmed that the large conformational changes are realized by the motions only in the flexible regions that connect CRDs. The Ca(2+) -ATPase efficiently utilizes its intrinsic flexibility and rigidity to response different switches like ligand binding/dissociation or ATP hydrolysis. The analysis detects functional motions without extensive biological knowledge of experts, suggesting its general applicability to domain movements in other membrane proteins to deepen the understanding of protein structure and function.

Cryo-electron microscopy has been used to visualize elongation factor G (EF-G) on the 70S ribosome in GDP and GTP states. GTP hydrolysis is required for binding of all the domains of EF-G to the pretranslocational complex and for the completion of translocation. In addition, large conformational changes have been identified in the ribosome. The head of the 30S subunit shifts toward the L1 protein side, and the L7/L12 stalk becomes bifurcated upon EF-G binding. Upon GTP hydrolysis, the bifurcation is reversed and an arc-like connection is formed between the base of the stalk and EF-G.

CG-COA: a coarse-grained cubic orientation approach for systematic protein-protein docking validation.

Plasminogen is known to undergo an extremely large conformational change when it binds ligands; the two well-established conformations are either closed (absence of external ligand) or open (presence of external ligand). We show here that plasminogen is more complicated than can be accommodated by a two-state, closed/open, model. Temperature changes induce large structural changes which can be detected with either dynamic light scattering or analytical ultracentrifugation. The temperature-induced changes are not related to the classical closed/open conformational change since both closed and open forms of the protein are similarly influenced. It appears as though the packing density of the protein increases as the temperature is raised. Over the range 4-20 degrees C, the Stokes' radius of the classical closed plasminogen goes from 4.7 to 4.2 nm, and that of the classical open form goes from 5.55 to 5.0 nm. These changes in packing can be rationalized if temperature change induces a large conformational change and if this is accompanied by a large change in hydration, by a change in solute binding, or by a change in the total void volume of the protein.

Agonist-Induced Conformational Changes in Bovine Rhodopsin: Insight into Activation of G-Protein-Coupled Receptors

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions