X-ray structure refinement and comparison of three forms of mitochondrial aspartate aminotransferase.

The X-ray crystal structures of three forms of the enzyme aspartate aminotransferase (EC 2.6.1.1) from chicken heart mitochondria have been refined by least-squares methods: holoenzyme with the co-factor pyridoxal-5'-phosphate bound at pH 7.5 (1.9 A resolution), holoenzyme with pyridoxal-5'-phosphate bound at pH 5.1 (2.3 A resolution) and holoenzyme with the co-factor pyridoxamine-5'-phosphate bound at pH 7.5 (2.2 A resolution). The crystallographic agreement factors [formula: see text] for the structures are 0.166, 0.130 and 0.131, respectively, for all data in the resolution range from 10.0 A to the limit of diffraction for each structure. The secondary, super-secondary and domain structures of the pyridoxal-phosphate holoenzyme at pH 7.5 are described in detail. The surface area of the interface between the monomer subunits of this dimeric alpha 2 protein is unusually large, indicating a very stable dimer. This is consistent with biochemical data. Both subunit and domain interfaces are relatively smooth compared with other proteins. The interactions of the protein with its co-factor are described and compared among the three structures. Observed changes in co-factor conformation may be related to spectral changes and the energetics of the catalytic reaction. Small but significant adjustments of the protein to changes in co-factor conformation are seen. These adjustments may be accommodated by small rigid-body shifts of secondary structural elements, and by packing defects in the protein core.     

Resolution-by-proxy: a simple measure for assessing and comparing the overall quality of NMR protein structures

In protein X-ray crystallography, resolution is often used as a good indicator of structural quality. Diffraction resolution of protein crystals correlates well with the number of X-ray observables that are used in structure generation and, therefore, with protein coordinate errors. In protein NMR, there is no parameter identical to X-ray resolution. Instead, resolution is often used as a synonym of NMR model quality. Resolution of NMR structures is often deduced from ensemble precision, torsion angle normality and number of distance restraints per residue. The lack of common techniques to assess the resolution of X-ray and NMR structures complicates the comparison of structures solved by these two methods. This problem is sometimes approached by calculating "equivalent resolution" from structure quality metrics. However, existing protocols do not offer a comprehensive assessment of protein structure as they calculate equivalent resolution from a relatively small number (<5) of protein parameters. Here, we report a development of a protocol that calculates equivalent resolution from 25 measurable protein features. This new method offers better performance (correlation coefficient of 0.92, mean absolute error of 0.28 ?) than existing predictors of equivalent resolution. Because the method uses coordinate data as a proxy for X-ray diffraction data, we call this measure "Resolution-by-Proxy" or ResProx. We demonstrate that ResProx can be used to identify under-restrained, poorly refined or inaccurate NMR structures, and can discover structural defects that the other equivalent resolution methods cannot detect. The ResProx web server is available at http://www.resprox.ca.     

Structure determination of secondary transport proteins by electron crystallography: two-dimensional crystallization of the betaine uptake system BetP

Structure determination at high resolution is still a challenge for membrane proteins in general, but in particular for secondary transporters due to their highly dynamic nature. X-ray structures of ten secondary transporters have recently been determined, but a thorough understanding of transport mechanisms necessitates structures at different functional states. Electron cryo-microscopy of two-dimensional (2D) crystals offers an alternative to obtain structural information at intermediate resolution. Electron crystallography is a sophisticated way to study proteins in a natural membrane environment and to track conformational changes in situ. Furthermore, basic interactions between protein and lipids can be investigated. Projection and 3-dimensional maps of six secondary transporters from different families have been determined by electron crystallography of 2D crystals at a resolution of 8 A and better. In this review, we give an overview about the principles of 2D crystallization, in particular of secondary transporters, and summarize the important steps successfully applied to establish and improve the 2D crystallization of the high-affinity glycine betaine uptake system from Corynebacterium glutamicum, BetP.     

Integrin Engagement by the Helical RGD Motif of the Helicobacter pylori CagL Protein Is Regulated by pH-induced Displacement of a Neighboring Helix

Arginine-aspartate-glycine (RGD) motifs are recognized by integrins to bridge cells to one another and the extracellular matrix. RGD motifs typically reside in exposed loop conformations. X-ray crystal structures of the Helicobacter pylori protein CagL revealed that RGD motifs can also exist in helical regions of proteins. Interactions between CagL and host gastric epithelial cell via integrins are required for the translocation of the bacterial oncoprotein CagA. Here, we have investigated the molecular basis of the CagL-host cell interactions using structural, biophysical, and functional analyses. We solved an x-ray crystal structure of CagL that revealed conformational changes induced by low pH not present in previous structures. Using analytical ultracentrifugation, we found that pH-induced conformational changes in CagL occur in solution and not just in the crystalline environment. By designing numerous CagL mutants based on all available crystal structures, we probed the functional roles of CagL conformational changes on cell surface integrin engagement. Together, our data indicate that the helical RGD motif in CagL is buried by a neighboring helix at low pH to inhibit CagL binding to integrin, whereas at neutral pH the neighboring helix is displaced to allow integrin access to the CagL RGD motif. This novel molecular mechanism of regulating integrin-RGD motif interactions by changes in the chemical environment provides new insight to H. pylori-mediated oncogenesis.     

The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid

The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.     

Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy

The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.     

Gorgon and pathwalking: macromolecular modeling tools for subnanometer resolution density maps

The complex interplay of proteins and other molecules, often in the form of large transitory assemblies, are critical to cellular function. Today, X-ray crystallography and electron cryo-microscopy (cryo-EM) are routinely used to image these macromolecular complexes, though often at limited resolutions. Despite the rapidly growing number of macromolecular structures, few tools exist for modeling and annotating structures in the range of 3-10 ? resolution. To address this need, we have developed a number of utilities specifically targeting subnanometer resolution density maps. As part of the 2010 Cryo-EM Modeling Challenge, we demonstrated two of our latest de novo modeling tools, Pathwalking and Gorgon, as well as a tool for secondary structure identification (SSEHunter) and a new rigid-body/flexible fitting tool in Gorgon. In total, we submitted 30 structural models from ten different subnanometer resolution data sets in four of the six challenge categories. Each of our utlities produced accurate structural models and annotations across the various density maps. In the end, the utilities that we present here offer users a robust toolkit for analyzing and modeling protein structure in macromolecular assemblies at non-atomic resolutions.     

A comparative analysis of 23 structures of the amyloidogenic protein transthyretin

Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.     

The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c-550 from Paracoccus versutus assessed by X-ray crystallography and unfolding

The structure of cytochrome c-550 from the nonphotosynthetic bacteria Paraccocus versutus has been solved by X-ray crystallography to 1.90 A resolution, and reveals a high structural homology to other bacterial cytochromes c(2). The effect of replacing the axial heme-iron methionine ligand with a lysine residue on protein structure and unfolding has been assessed using the M100K variant. From X-ray structures at 1.95 and 1.55 A resolution it became clear that the amino group of the lysine side chain coordinates to the heme-iron. Structural differences compared to the wild-type protein are confined to the lysine ligand loop connecting helices four and five. In the heme cavity an additional water molecule is found which participates in an H-bonding interaction with the lysine ligand. Under cryo-conditions extra electron density in the lysine ligand loop is revealed, leading to residues K97 to T101 being modeled with a double main-chain conformation. Upon unfolding, dissociation of the lysine ligand from the heme-iron is shown to be pH dependent, with NMR data consistent with the occurrence of a ligand exchange mechanism similar to that seen for the wild-type protein.     

Structure of cubic insulin crystals in glucose solutions.

X-ray structures of cubic insulin crystals in high concentrations of glucose at different pH levels and temperatures have been refined to high resolution. We have identified one glucose-binding site near the N-terminus of the A-chain whose occupancy is pH dependent. The effects of reduced water activity on the ordered protein and solvent structures have been examined. Our analysis showed no notable conformational changes in the ordered protein structures or ordered solvent molecules near the protein surface, but the presence of glucose does have a significant effect on the overall density distribution of the bulk solvent in the solvent-accessible volume. We compared the structure of cubic insulin at room temperature and liquid-nitrogen temperature, under identical solvent conditions, using glucose as a cryoprotectant. In this case, we found that the average temperature factor of the protein is reduced and more water molecules can be identified, but there are no significant changes in the protein conformation.     

High-resolution structures of retinol-binding protein in complex with retinol: pH-induced protein structural changes in the crystal state

The targeted delivery of non-polar ligands by binding proteins to membranes or membrane receptors involves the release of these ligands on or near the plasma membrane of target cells. Because these hydrophobic ligands are often bound inside a deep cavity of binding proteins, as shown previously for plasma retinol-binding protein (RBP), their release from these proteins might require the destabilization of the protein structure by partially denaturing conditions, such as those possibly present near plasma membranes. RBP is a plasma transport protein which delivers specifically retinol from its store sites to target cells. Here, we report the high-resolution (1.1-1.4A) crystal structures of bovine holo-RBP at five different pH values, ranging from 9 to 2. While unraveling details of the native protein structure and of the interactions with retinol at nearly atomic resolution at neutral pH, this study provides evidence for definite pH-induced modifications of several structural features of RBP. The structure most representative of the changes that holo-RBP undergoes at different pH values is that of its flexible state at pH 2. At this pH, most significant are the alteration of the arrangement of salt bridges and of the network of water molecules/H-bonds that participates in the retinol-RBP interaction, an appreciable increase of the volume of the beta-barrel cavity, a considerably higher degree of mobility of the RBP-bound ligand and of several protein regions and the disorder of a large number of solvent molecules that are ordered at neutral pH. These changes are likely to be accompanied by a modification of the pattern of charge distribution on the protein surface. All these changes, which reveal a substantially lowered conformational stability of RBP, presumably occur at the initial stages of the acidic denaturation of RBP and are possibly associated with a facilitated release of the retinol molecule from its carrier protein.     

Structural origins of pH‐dependent chemical shifts in the B1 domain of protein G

We report chemical shifts for H^N, N, and C′ nuclei in the His‐tagged B1 domain of protein G (GB1) over a range of pH values from pH 2.0 to 9.0, which fit well to standard pH‐dependent equations. We also report a 1.2 Å resolution crystal structure of GB1 at pH 3.0. Comparison of this crystal structure with published crystal structures at higher pHs provides details of the structural changes in GB1 associated with protonation of the carboxylate groups, in particular a conformational change in the C‐terminus of the protein at low pH. An additional change described recently is not seen in the crystal structure because of crystal contacts. We show that the pH‐dependent changes in chemical shifts can be almost entirely understood based on structural changes, thereby providing insight into the relationship between structure and chemical shift. In particular, we describe through‐bond effects extending up to five bonds, affecting N and C′ but not H^N; through‐space effects of carboxylates, which fit well to a simple electric field model; and effects due to conformational change, which have a similar magnitude to many of the direct effects. Finally, we discuss cooperative effects, demonstrating a lack of cooperative unfolding in the helix, and the existence of a β‐sheet “iceberg” extending over three of the four strands. This study therefore extends the application of chemical shifts to understanding protein structure. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Constructing and validating initial Cα models from subnanometer resolution density maps with pathwalking

A significant number of macromolecular structures solved by electron cryo-microscopy and X-ray crystallography obtain resolutions of 3.5-6?, at which direct atomistic interpretation is difficult. To address this, we developed pathwalking, a semi-automated protocol to enumerate reasonable Cα models from near-atomic resolution density maps without a structural template or sequence-structure correspondence. Pathwalking uses an approach derived from the Traveling Salesman Problem to rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models. Pathwalking can also be used to validate and identify potential structural ambiguities in models generated from near-atomic resolution density maps. In this work, examples from the EMDB and PDB are used to assess the broad applicability and accuracy of our method. With the growing number of near-atomic resolution density maps from cryo-EM and X-ray crystallography, pathwalking can become an important tool in modeling protein structures.     

Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements

Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site.     

Structural basis of affinity maturation and intramolecular cooperativity in a protein-protein interaction

Although protein-protein interactions are involved in nearly all cellular processes, general rules for describing affinity and selectivity in protein-protein complexes are lacking, primarily because correlations between changes in protein structure and binding energetics have not been well determined. Here, we establish the structural basis of affinity maturation for a protein-protein interaction system that we had previously characterized energetically. This model system exhibits a 1500-fold affinity increase. Also, its affinity maturation is restricted by negative intramolecular cooperativity. With three complex and six unliganded variant X-ray crystal structures, we provide molecular snapshots of protein interface remodeling events that span the breadth of the affinity maturation process and present a comprehensive structural view of affinity maturation. Correlating crystallographically observed structural changes with measured energetic changes reveals molecular bases for affinity maturation, intramolecular cooperativity, and context-dependent binding.     

Effects of environment on the structure of Pyrococcus furiosus rubredoxin: a molecular dynamics study

Molecular dynamics simulations based on a 0.95-A resolution crystal structure of Pyrococcus furiosus have been performed to elucidate the effects of the environment on the structure of rubredoxin, and proteins in general. Three 1-ns simulations are reported here: two crystalline state simulations at 123 and 300 K, and a solution state simulation at 300 K. These simulations show that temperature has a greater impact on the protein structure than the close molecular contacts of the crystal matrix in rubredoxin, although both have an effect on its dynamic properties. These results indicate that differences between NMR solution structures and X-ray crystal structures will be relatively minor if they are done at similar temperatures. In addition, the crystal simulations appears to mimic previous crystallographic experiments on the effects of cryo-temperature on temperature factors, and might provide a useful tool in the structural analysis of protein structures solved at cryo-temperatures.     

Structural characterization of an early fusion intermediate of influenza virus hemagglutinin

The hemagglutinin (HA) envelope protein of influenza virus mediates viral entry through membrane fusion in the acidic environment of the endosome. Crystal structures of HA in pre- and postfusion states have laid the foundation for proposals for a general fusion mechanism for viral envelope proteins. The large-scale conformational rearrangement of HA at low pH is triggered by a loop-to-helix transition of an interhelical loop (B loop) within the fusion domain and is often referred to as the "spring-loaded" mechanism. Although the receptor-binding HA1 subunit is believed to act as a "clamp" to keep the B loop in its metastable prefusion state at neutral pH, the "pH sensors" that are responsible for the clamp release and the ensuing structural transitions have remained elusive. Here we identify a mutation in the HA2 fusion domain from the influenza virus H2 subtype that stabilizes the HA trimer in a prefusion-like state at and below fusogenic pH. Crystal structures of this putative early intermediate state reveal reorganization of ionic interactions at the HA1-HA2 interface at acidic pH and deformation of the HA1 membrane-distal domain. Along with neutralization of glutamate residues on the B loop, these changes cause a rotation of the B loop and solvent exposure of conserved phenylalanines, which are key residues at the trimer interface of the postfusion structure. Thus, our study reveals the possible initial structural event that leads to release of the B loop from its prefusion conformation, which is aided by unexpected structural changes within the membrane-distal HA1 domain at low pH.     

Conformational Changes and Ligand Recognition of Escherichia colid-Xylose Binding Protein Revealed

ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-ray structures of d-xylose binding protein from Escherichia coli in ligand-free open form, ligand-bound open form, and ligand-bound closed form at 2.15 Å, 2.2 Å, and 2.2 Å resolutions, respectively. The ligand-bound open form is the first such structure to be reported at high resolution; the combination of the three different forms from the same protein furthermore gives unprecedented details concerning the conformational changes involved in binding protein function. As is typical of the structural family, the protein has two similar globular domains, which are connected by a three-stranded hinge region. The open liganded structure shows that xylose binds first to the C-terminal domain, with only very small conformational changes resulting. After a 34° closing motion, additional interactions are formed with the N-terminal domain; changes in this domain are larger and serve to make the structure more ordered near the ligand. An analysis of the interactions suggests why xylose is the preferred ligand. Furthermore, a comparison with the most closely related proteins in the structural family shows that the conformational changes are distinct in each type of binding protein, which may have implications for how the individual proteins act in concert with their respective membrane permeases.     

Knowledge-based interaction potentials for proteins.

We discuss the derivation of atomic-level potentials of mean force from the known protein structures and their applicability for structural evaluation applications. In the derivation process, rigorous density estimation methodology is used to estimate the probability density functions (PDFs) for the distributions of interatomic distances in the protein structures. Potentials of mean force are then derived from these density functions using simple Boltzmann's relation. We also test the potentials against pairs of current and superseded protein structures in the Protein Data Bank. Using PDF potentials to evaluate each structure pair, we are able to identify, with high accuracy, which of the two structures is of higher resolution or better quality. This result shows that the PDF potentials are sensitive to details in protein structures as the current and superseded atomic coordinates generally do not differ by more than 1 A in root-mean-square deviation, and that the PDF potentials could potentially be used for X-ray structure refinement and protein structure prediction.