Critical evaluation of a crystal structure of nitrogenase with bound N2 ligands.

Recently, a 1.83 Å crystallographic structure of nitrogenase was suggested to show N2-derived ligands at three sites in the catalytic FeMo cluster, replacing the three [Formula: see text] bridging sulfide ligands (two in one subunit and the third in the other subunit) (Kang et al. in Science 368: 1381-1385, 2020). Naturally, such a structure is sensational, having strong bearings on the reaction mechanism of the enzyme. Therefore, it is highly important to ensure that the interpretation of the structure is correct. Here, we use standard crystallographic refinement and quantum refinement to evaluate the structure. We show that the original crystallographic raw data are strongly anisotropic, with a much lower resolution in certain directions than others. This, together with the questionable use of anisotropic B factors, give atoms an elongated shape, which may look like diatomic atoms. In terms of standard electron-density maps and real-space Z scores, a resting-state structure with no dissociated sulfide ligands fits the raw data better than the interpretation suggested by the crystallographers. The anomalous electron density at 7100 eV is weaker for the putative N2 ligands, but not lower than for several of the [Formula: see text] bridging sulfide ions and not lower than what can be expected from a statistical analysis of the densities. Therefore, we find no convincing evidence for any N2 binding to the FeMo cluster. Instead, a standard resting state without any dissociated ligands seems to be the most likely interpretation of the structure. Likewise, we find no support that the homocitrate ligand should show monodentate binding.

A protocol for production of perdeuterated OmpF porin for neutron crystallography.

Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling - perdeuteration - requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.

Interplay between Conformational Entropy and Solvation Entropy in Protein-Ligand Binding.

Understanding the driving forces underlying molecular recognition is of fundamental importance in chemistry and biology. The challenge is to unravel the binding thermodynamics into separate contributions and to interpret these in molecular terms. Entropic contributions to the free energy of binding are particularly difficult to assess in this regard. Here we pinpoint the molecular determinants underlying differences in ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and molecular dynamics simulations followed by conformational entropy and grid inhomogeneous solvation theory (GIST) analyses. Using a pair of diastereomeric ligands that have essentially identical chemical potential in the unbound state, we reduced the problem of dissecting the thermodynamics to a comparison of the two protein-ligand complexes. While the free energies of binding are nearly equal for the R and S diastereomers, greater differences are observed for the enthalpy and entropy, which consequently exhibit compensatory behavior, ΔΔ H°(R - S) = -5 ± 1 kJ/mol and - TΔΔ S°(R - S) = 3 ± 1 kJ/mol. NMR relaxation experiments and molecular dynamics simulations indicate that the protein in complex with the S-stereoisomer has greater conformational entropy than in the R-complex. GIST calculations reveal additional, but smaller, contributions from solvation entropy, again in favor of the S-complex. Thus, conformational entropy apparently dominates over solvation entropy in dictating the difference in the overall entropy of binding. This case highlights an interplay between conformational entropy and solvation entropy, pointing to both opportunities and challenges in drug design.

Are crystallographic B-factors suitable for calculating protein conformational entropy?

Conformational entropies are of great interest when studying the binding of small ligands to proteins or the interaction of proteins. Unfortunately, there are no experimental methods available to measure conformational entropies of all groups in a protein. Instead, they are normally estimated from molecular dynamics (MD) simulations, although such methods show problems with convergence and correlation of motions, and depend on the accuracy of the underlying potential-energy function. Crystallographic atomic displacement parameters (also known as B-factors) are available in all crystal structures and contain information about the atomic fluctuations, which can be converted to entropies. We have studied whether B-factors can be employed to extract conformational entropies for proteins by comparing such entropies to those measured by NMR relaxation experiments or obtained from MD simulations in solution or in the crystal. Unfortunately, our results show that B-factor entropies are unreliable, because they include the movement and rotation of the entire protein, they exclude correlation of the movements and they include contributions other than the fluctuations, e.g. static disorder, as well as errors in the model and the scattering factors. We have tried to reduce the first problem by employing translation-libration-screw refinement, the second by employing a description of the correlated movement from MD simulations, and the third by studying only the change in entropy when a pair of ligands binds to the same protein, thoroughly re-refining the structures in exactly the same way and using the same set of alternative conformations. However, the experimental B-factors seem to be incompatible with fluctuations from MD simulations and the precision is too poor to give any reliable entropies.

Neutron Crystallography for the Study of Hydrogen Bonds in Macromolecules.

Abstract: The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, the protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. This article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.

Is the bovine lysosomal phospholipase B-like protein an amidase?

The main function of lysosomal proteins is to degrade cellular macromolecules. We purified a novel lysosomal protein to homogeneity from bovine kidneys. By gene annotation, this protein is defined as a bovine phospholipase B-like protein 1 (bPLBD1) and, to better understand its biological function, we solved its structure at 1.9 Å resolution. We showed that bPLBD1 has uniform noncomplex-type N-glycosylation and that it localized to the lysosome. The first step in lysosomal protein transport, the initiation of mannose-6-phosphorylation by a N-acetylglucosamine-1-phosphotransferase, requires recognition of at least two distinct lysines on the protein surface. We identified candidate lysines by analyzing the structural and sequentially conserved N-glycosylation sites and lysines in bPLBD1 and in the homologous mouse PLBD2. Our model suggests that N408 is the primarily phosphorylated glycan, and K358 a key residue for N-acetylglucosamine-1-phosphotransferase recognition. Two other lysines, K334 and K342, provide the required second site for N-acetylglucosamine-1-phosphotransferase recognition. bPLBD1 is an N-terminal nucleophile (Ntn) hydrolase. By comparison with other Ntn-hydrolases, we conclude that the acyl moiety of PLBD1 substrate must be small to fit the putative binding pocket, whereas the space for the rest of the substrate is a large open cleft. Finally, as all the known substrates of Ntn-hydrolases have amide bonds, we suggest that bPLBD1 may be an amidase or peptidase instead of lipase, explaining the difficulty in finding a good substrate for any members of the PLBD family.

The structure of the NTPase that powers DNA packaging into Sulfolobus turreted icosahedral virus 2.

Biochemical reactions powered by ATP hydrolysis are fundamental for the movement of molecules and cellular structures. One such reaction is the encapsidation of the double-stranded DNA (dsDNA) genome of an icosahedrally symmetric virus into a preformed procapsid with the help of a genome-translocating NTPase. Such NTPases have been characterized in detail from both RNA and tailed DNA viruses. We present four crystal structures and the biochemical activity of a thermophilic NTPase, B204, from the nontailed, membrane-containing, hyperthermoacidophilic archaeal dsDNA virus Sulfolobus turreted icosahedral virus 2. These are the first structures of a genome-packaging NTPase from a nontailed, dsDNA virus with an archaeal host. The four structures highlight the catalytic cycle of B204, pinpointing the molecular movement between substrate-bound (open) and empty (closed) active sites. The protein is shown to bind both single-stranded and double-stranded nucleic acids and to have an optimum activity at 80°C and pH 4.5. The overall fold of B204 places it in the FtsK-HerA superfamily of P-loop ATPases, whose cellular and viral members have been suggested to share a DNA-translocating mechanism.

A combined quantum chemical and crystallographic study on the oxidized binuclear center of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. By reducing oxygen to water, it generates a proton gradient across the mitochondrial or bacterial membrane. Recently, two independent X-ray crystallographic studies ((Aoyama et al. Proc. Natl. Acad. Sci. USA 106 (2009) 2165-2169) and (Koepke et al. Biochim. Biophys. Acta 1787 (2009) 635-645)), suggested that a peroxide dianion might be bound to the active site of oxidized CcO. We have investigated this hypothesis by combining quantum chemical calculations with a re-refinement of the X-ray crystallographic data and optical spectroscopic measurements. Our data suggest that dianionic peroxide, superoxide, and dioxygen all form a similar superoxide species when inserted into a fully oxidized ferric/cupric binuclear site (BNC). We argue that stable peroxides are unlikely to be confined within the oxidized BNC since that would be expected to lead to bond splitting and formation of the catalytic P intermediate. Somewhat surprisingly, we find that binding of dioxygen to the oxidized binuclear site is weakly exergonic, and hence, the observed structure might have resulted from dioxygen itself or from superoxide generated from O(2) by the X-ray beam. We show that the presence of O(2) is consistent with the X-ray data. We also discuss how other structures, such as a mixture of the aqueous species (H(2)O+OH(-) and H(2)O) and chloride fit the experimental data.

Combining crystallography with quantum mechanics.

In standard crystallographic refinement of biomacromolecules, the crystallographic raw data are supplemented by empirical restraints that ensure that the structure makes chemical sense. These restraints are typically accurate for amino acids and nucleic acids, but less so for cofactors, substrates, inhibitors, ligands and metal sites. In quantum refinement, this potential is replaced by more accurate quantum mechanical (QM) calculations. Several implementations have been presented, differing in the level of QM and whether it is used for the entire structure or only for a site of particular interest. It has been shown that the method can improve and correct errors in crystal structures and that it can be used to determine protonation and tautomeric states of various ligands and to decide what is really seen in the structure by refining different interpretations and using standard crystallographic and QM quality measures to decide which fits the structure best.

Quantum-refinement studies of the bidentate ligand of V­nitrogenase and the protonation state of CO-inhibited Mo­nitrogenase.

Nitrogenase is the only enzyme that can cleave the triple bond in N2, making nitrogen available to plants (although the enzyme itself is strictly microbial). It has been studied extensively with both experimental and computational methods, but many details of the reaction mechanism are still unclear. X-ray crystallography is the main source of structural information for biomacromolecules, but it has problems to discern hydrogen atoms or to distinguish between elements with the same number of electrons. These problems can sometimes be alleviated by introducing quantum chemical calculations in the refinement, providing information about the ideal structure (in the same way as the empirical restraints used in standard crystallographic refinement) and comparing different interpretations of the structure with normal crystallographic and quantum mechanical quality measures. We have performed such quantum-refinement calculations to address two important issues for nitrogenase. First, we show that the bidentate ligand of the active-site FeV cluster in V­nitrogenase is carbonate, rather than bicarbonate or nitrate. Second, we study the CO-inhibited structure of Mo­nitrogenase. CO binds to a reduced and protonated state of the enzyme by replacing one of the sulfide ions (S2B) in the active-site FeMo cluster. We examined if it is possible to deduce from the crystal structure the location of the protons. Our results indicates that the crystal structure is best modelled as fully deprotonated.

Exploring ligand dynamics in protein crystal structures with ensemble refinement.

Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein-ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein-ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and Rfree values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.

Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps.

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from its ability to facilitate the deprotonation of a carbon next to a carbonyl group to generate an enediolate intermediate. The enediolate intermediate is believed to be mimicked by the inhibitor 2-phosphoglycolate (PGA) and the subsequent enediol intermediate by phosphoglycolohydroxamate (PGH). Here, neutron structures of Leishmania mexicana TIM have been determined with both inhibitors, and joint neutron/X-ray refinement followed by quantum refinement has been performed. The structures show that in the PGA complex the postulated general base Glu167 is protonated, while in the PGH complex it remains deprotonated. The deuteron is clearly localized on Glu167 in the PGA-TIM structure, suggesting an asymmetric hydrogen bond instead of a low-barrier hydrogen bond. The full picture of the active-site protonation states allowed an investigation of the reaction mechanism using density-functional theory calculations.

Entropy-Entropy Compensation between the Protein, Ligand, and Solvent Degrees of Freedom Fine-Tunes Affinity in Ligand Binding to Galectin-3C.

Molecular recognition is fundamental to biological signaling. A central question is how individual interactions between molecular moieties affect the thermodynamics of ligand binding to proteins and how these effects might propagate beyond the immediate neighborhood of the binding site. Here, we investigate this question by introducing minor changes in ligand structure and characterizing the effects of these on ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and computational approaches including molecular dynamics (MD) simulations and grid inhomogeneous solvation theory (GIST). We studied a congeneric series of ligands with a fluorophenyl-triazole moiety, where the fluorine substituent varies between the ortho, meta, and para positions (denoted O, M, and P). The M and P ligands have similar affinities, whereas the O ligand has 3-fold lower affinity, reflecting differences in binding enthalpy and entropy. The results reveal surprising differences in conformational and solvation entropy among the three complexes. NMR backbone order parameters show that the O-bound protein has reduced conformational entropy compared to the M and P complexes. By contrast, the bound ligand is more flexible in the O complex, as determined by 19F NMR relaxation, ensemble-refined X-ray diffraction data, and MD simulations. Furthermore, GIST calculations indicate that the O-bound complex has less unfavorable solvation entropy compared to the other two complexes. Thus, the results indicate compensatory effects from ligand conformational entropy and water entropy, on the one hand, and protein conformational entropy, on the other hand. Taken together, these different contributions amount to entropy-entropy compensation among the system components involved in ligand binding to a target protein.

Crystallization and preliminary crystallographic analysis of mouse peroxiredoxin II with significant pseudosymmetry.

Peroxiredoxin II was cloned from mouse B cells into pCold 1 expression vector and produced as a His-tagged recombinant protein in Escherichia coli. A ring form was isolated by gel filtration. A crystal obtained by the sitting-drop vapour-diffusion method diffracted to 1.77 A resolution at 100 K. The crystal belonged to space group P2(1)2(1)2, with unit-cell parameters a = 117.4, b = 133.9, c = 139.1 A. The asymmetric unit is expected to contain six dimers of peroxiredoxin II, with a corresponding solvent content of 39.3%. Peaks in the native Patterson function together with pseudo-systematic absences suggested that the crystals suffered from severe translational pseudosymmetry.

Neutron macromolecular crystallography at the European spallation source.

The long-pulse spallation source of the European Spallation Source-a facility under construction in Lund, Sweden-is well suited for macromolecular crystallography experiments. We review briefly the particular properties of the long-pulse source and the associated high-brilliance moderators from the point of view of instrument design. We then outline the design philosophy and current design of the NMX macromolecular diffractometer. We also briefly describe the supporting facilities available for users and finish by an evaluation of the expected performance.

Automated orientation of water molecules in neutron crystallographic structures of proteins.

The structure and function of proteins are strongly affected by the surrounding solvent water, for example through hydrogen bonds and the hydrophobic effect. These interactions depend not only on the position, but also on the orientation, of the water molecules around the protein. Therefore, it is often vital to know the detailed orientations of the surrounding ordered water molecules. Such information can be obtained by neutron crystallography. However, it is tedious and time-consuming to determine the correct orientation of every water molecule in a structure (there are typically several hundred of them), which is presently performed by manual evaluation. Here, a method has been developed that reliably automates the orientation of a water molecules in a simple and relatively fast way. Firstly, a quantitative quality measure, the real-space correlation coefficient, was selected, together with a threshold that allows the identification of water molecules that are oriented. Secondly, the refinement procedure was optimized by varying the refinement method and parameters, thus finding settings that yielded the best results in terms of time and performance. It turned out to be favourable to employ only the neutron data and a fixed protein structure when reorienting the water molecules. Thirdly, a method has been developed that identifies and reorients inadequately oriented water molecules systematically and automatically. The method has been tested on three proteins, galectin-3C, rubredoxin and inorganic pyrophosphatase, and it is shown that it yields improved orientations of the water molecules for all three proteins in a shorter time than manual model building. It also led to an increased number of hydrogen bonds involving water molecules for all proteins.

fragHAR: towards ab initio quantum-crystallographic X-ray structure refinement for polypeptides and proteins.

The first ab initio aspherical structure refinement against experimental X-ray structure factors for polypeptides and proteins using a fragmentation approach to break up the protein into residues and solvent, thereby speeding up quantum-crystallographic Hirshfeld atom refinement (HAR) calculations, is described. It it found that the geometric and atomic displacement parameters from the new fragHAR method are essentially unchanged from a HAR on the complete unfragmented system when tested on dipeptides, tripeptides and hexapeptides. The largest changes are for the parameters describing H atoms involved in hydrogen-bond interactions, but it is shown that these discrepancies can be removed by including the interacting fragments as a single larger fragment in the fragmentation scheme. Significant speed-ups are observed for the larger systems. Using this approach, it is possible to perform a highly parallelized HAR in reasonable times for large systems. The method has been implemented in the TONTO software.

Water structure in solution and crystal molecular dynamics simulations compared to protein crystal structures.

The function of proteins is influenced not only by the atomic structure but also by the detailed structure of the solvent surrounding it. Computational studies of protein structure also critically depend on the water structure around the protein. Herein we compare the water structure obtained from molecular dynamics (MD) simulations of galectin-3 in complex with two ligands to crystallographic water molecules observed in the corresponding crystal structures. We computed MD trajectories both in a water box, which mimics a protein in solution, and in a crystallographic unit cell, which mimics a protein in a crystal. The calculations were compared to crystal structures obtained at both cryogenic and room temperature. Two types of analyses of the MD simulations were performed. First, the positions of the crystallographic water molecules were compared to peaks in the MD density after alignment of the protein in each snapshot. The results of this analysis indicate that all simulations reproduce the crystallographic water structure rather poorly. However, if we define the crystallographic water sites based on their distances to nearby protein atoms and follow these sites throughout the simulations, the MD simulations reproduce the crystallographic water sites much better. This shows that the failure of MD simulations to reproduce the water structure around proteins in crystal structures observed both in this and previous studies is caused by the problem of identifying water sites for a flexible and dynamic protein (traditionally done by overlaying the structures). Our local clustering approach solves the problem and shows that the MD simulations reasonably reproduce the water structure observed in crystals. Furthermore, analysis of the crystal MD simulations indicates a few water molecules that are close to unmodeled electron density peaks in the crystal structures, suggesting that crystal MD could be used as a complementary tool for identifying and modelling water in protein crystallography.

Refinement of protein structures using a combination of quantum-mechanical calculations with neutron and X-ray crystallographic data. Corrigendum.

Corrections are published for the article by Caldararu et al. [(2019), Acta Cryst. D75, 368-380].

Prospects for membrane protein crystals in NMX.

Adding hydrogen atoms and protonation states to structures of membrane proteins requires successful implementation of neutron macromolecular crystallography (NMX). This information would significantly increase our fundamental understanding of the transport processes membrane proteins undertake. To grow the large crystals needed for NMX studies requires significant amounts of stable protein, but once that challenge is overcome there is no intrinsic property of membrane proteins preventing the growth of large crystals per se. The calcium-transporting P-type ATPase (SERCA) has been thoroughly characterized biochemically and structurally over decades. We have extended our crystallization efforts to assess the feasibility of growing SERCA crystals for NMX-exploring microdialysis and capillary counterdiffusion crystallization techniques as alternatives to the traditional vapor diffusion crystallization experiment. Both methods possess crystallization dynamics favorable for maximizing crystal size and we used them to facilitate the growth of large crystals, validating these approaches for membrane protein crystallization for NMX.