Subatomic and atomic crystallographic studies of aldose reductase: implications for inhibitor binding

The determination of several of aldose reductase-inhibitor complexes at subatomic resolution has revealed new structural details, including the specific interatomic contacts involved in inhibitor binding. In this article, we review the structures of the complexes of ALR2 with IDD 594 (resolution: 0.66 Å, IC50 (concentration of the inhibitor that produced half-maximal effect): 30 nM, space group: P2¹), IDD 393 (resolution: 0.90 Å, IC50: 6 nM, space group: P1), fidarestat (resolution: 0.92 Å, IC50: 9 nM, space group: P2¹) and minalrestat (resolution: 1.10 Å, IC50: 73 nM, space group: P1). The structures are compared and found to be highly reproductible within the same space group (root mean square (RMS) deviations: 0.15 0.3 Å). The mode of binding of the carboxylate inhibitors IDD 594 and IDD 393 is analysed. The binding of the carboxylate head can be accurately determined by the subatomic resolution structures, since both the protonation states and the positions of the atoms are very precisely known. The differences appear in the binding in the specificity pocket. The high-resolution structures explain the differences in IC50, which are confirmed both experimentally by mass spectrometry measures of VC50 and theoretically by free energy perturbation calculations. The binding of the cyclic imide inhibitors fidarestat and minalrestat is also described, focusing on the observation of a Cl^- ion which binds simultaneously with fidarestat. The presence of this anion, binding also to the active site residue His110, leads to a mechanism in which the inhibitor can bind in a neutral state and then become charged inside the active site pocket. This mechanism can explain the excellent in vivo properties of cyclic imide inhibitors. In summary, the complete and detailed information supplied by the subatomic resolution structures can explain the differences in binding energy of the different inhibitors.     

Three-dimensional structure of motor molecules

Images, calculated from electron micrographs, show the three-dimensional structures of microtubules and tubulin sheets decorated stoichiometrically with motor protein molecules. Dimeric motor domains (heads) of kinesin and ncd, the kinesin-related protein that moves in the reverse direction, each appeared to bind to tubulin in the same way, by one of their two heads. The second heads show an interesting difference in position that seems to be related to the directions of movement of the two motors. X-ray crystallographic results showing the structures of kinesin and ncd to be very similar at atomic resolution, and homologous also to myosin, suggest that the two motor families may use mechanisms that have much in common. Nevertheless, myosins and kinesins differ kinetically. Also, whereas conformational changes in the myosin catalytic domain are amplified by a long lever arm that connects it to the stalk domain, kinesin and ncd do not appear to possess a structure with a similar function but may rely on biased diffusion in order to move along microtubules.     

Protein surface similarities: a survey of methods to describe and compare protein surfaces

Many methods have been developed to analyse protein sequences and structures, although less work has been undertaken describing and comparing protein surfaces. Evolution can lead sequences to diverge or structures to change topology; nevertheless, surface determinants that are essential to protein function itself may be mantained. Moreover, different molecules could converge to similar functions by gaining specific surface determinants. In such cases, sequence or structure comparisons are likely to be inadequate in describing or identifying protein functions and evolutionary relationships among proteins. Surface analysis can identify function determinants that are independent of sequence or secondary structure and can therefore be a powerful tool to highlight cases of possible convergent or divergent evolution. This kind of approach can be useful for a better understanding of protein molecular and biochemical mechanisms of catalysis or interaction with a ligand, which are usually surface dependent. Protein surface comparison, when compared to sequence or structure comparison methods, is a hard computational challenge and evaluated methods allowing the comparison of protein surfaces are difficult to find. In this review, we will survey the current knowledge about protein surface similarity and the techniques to detect it.     

Ultrastructure of epiphyseal cartilage after extraction on thin sections of "Epon" of proteoglycans by calcium chloride]

Dissociative methods are commonly used to extract proteoglycans. With a 2 M CaCl2 solution these components can be also extracted from thin sections of fixed, "Epon" included material. Secretory granules of the chondrocytes, granular components of extracellular matrix as matrix vesicles lost their electron density. Glycoproteins of the cell coat as non collagenous glycoproteins disappear. The method seems to be valuable for extracting proteoglycans from thin sections prepared for electron microscope investigations.     

Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.     

The leucine-rich repeat structure

The leucine-rich repeat is a widespread structural motif of 20-30 amino acids with a characteristic repetitive sequence pattern rich in leucines. Leucine-rich repeat domains are built from tandems of two or more repeats and form curved solenoid structures that are particularly suitable for protein-protein interactions. Thousands of protein sequences containing leucine-rich repeats have been identified by automatic annotation methods. Three-dimensional structures of leucine-rich repeat domains determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. As the essential structural principles become well established, the leucine-rich repeat architecture is emerging as an attractive framework for structural prediction and protein engineering. This review presents an update of the current understanding of leucine-rich repeat structure at the primary, secondary, tertiary and quaternary levels and discusses specific examples from recently determined three-dimensional structures.     

Mining electron density for functionally relevant protein polysterism in crystal structures

This review focuses on conceptual and methodological advances in our understanding and characterization of the conformational heterogeneity of proteins. Focusing on X-ray crystallography, we describe how polysterism, the interconversion of pre-existing conformational substates, has traditionally been analyzed by comparing independent crystal structures or multiple chains within a single crystal asymmetric unit. In contrast, recent studies have focused on mining electron density maps to reveal previously ‘hidden’ minor conformational substates. Functional tests of the importance of minor states suggest that evolutionary selection shapes the entire conformational landscape, including uniquely configured conformational substates, the relative distribution of these substates, and the speed at which the protein can interconvert between them. An increased focus on polysterism may shape the way protein structure and function is studied in the coming years.     

Scanning transmission electron microscopy of dendritic spines stained by the Golgi method]

Scanning transmission electron microscopy of the dendritic spines of multipolar neurons in the cat inferior Colliculus was achieved on Golgi semi-thin sections. The three basic types of dendritic spines (ST, MS, TH) were identified. Scanning transmission electron microscopy provides a reliable method for a three dimensional view of these structures at high resolution and consequently a more accurate appreciation of their size. In addition, it could prove very useful in the quantitative analysis of the dendritic spines.     

A dynamic view of peptides and proteins in membranes

Biological membranes are highly dynamic supramolecular arrangements of lipids and proteins, which fulfill key cellular functions. Relatively few high-resolution membrane protein structures are known to date, although during recent years the structural databases have expanded at an accelerated pace. In some instances the structures of reaction intermediates provide a stroboscopic view on the conformational changes involved in protein function. Other biophysical approaches add dynamic aspects and allow one to investigate the interactions with the lipid bilayers. Membrane-active peptides fulfill many important functions in nature as they act as antimicrobials, channels, transporters or hormones, and their studies have much increased our understanding of polypeptide-membrane interactions. Interestingly several proteins have been identified that interact with the membrane as loose arrays of domains. Such conformations easily escape classical high-resolution structural analysis and the lessons learned from peptides may therefore be instructive for our understanding of the functioning of such membrane proteins. Received 11 March 2008; received after revision 2 May 2008; accepted 5 May 2008     

The interface of protein-protein complexes: Analysis of contacts and prediction of interactions

Specific protein-protein interactions are essential for cellular functions. Experimentally determined three-dimensional structures of protein-protein complexes offer the possibility to characterize binding interfaces in terms of size, shape and packing density. Comparison with crystal-packing interfaces representing nonspecific protein-protein contacts gives insight into how specific binding differs from nonspecific low-affinity binding. An overview is given on empirical structural rules for specific protein-protein recognition derived from known complex structures. Although single parameters such as interface size, shape or surface complementary show clear trends for different interface types, each parameter alone is insufficient to fully distinguish between specific versus crystal-packing contacts. A combination of interface parameters is, however, well suited to characterize a specific interface. This knowledge provides us with the essential ingredients that make up a specific protein recognition site. It is also of great value for the prediction of protein binding sites and for the evaluation of predicted complex structures. Received 1 October 2007; received after revision 9 November 2007; accepted 9 November 2007     

Perforin and its role in T lymphocyte-mediated cytolysis

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack.Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.     

Perforin and its role in T lymphocyte-mediated cytolysis.

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack. Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.     

Roles of postsynaptic density-95/synapse-associated protein 90 and its interacting proteins in the organization of synapses

Synapses are central stages for neurotransmission. Neurotransmitters are released from the presynaptic membrane of one neuron, and bind to the receptors accumulated at the postsynaptic membrane, followed by the activation of the other neuron. The strength of a synapse is modified depending on the history of the previous neurotransmissions. This property is called synaptic plasticity and is implicated in learning and memory. Synapses contain not only the components essential for neurotransmission but also the signalling molecules involved in synaptic plasticity. The elucidation of the molecular structures of synapses is one of the key steps to understand the mechanism of learning and memory. Recent studies have revealed postsynaptic density (PSD)-95/synapse-associated protein (SAP) 90 as a core component in the architecture of synapses. In this review, we summarize up-to-date information about PSD-95/SAP90 and its interacting proteins, and the organization of synapses orchestrated     

Benzodiazepine receptors resolved

Summary To date, attempts to map the distribution and density of benzodiazepine receptors in the CNS have been dominated by radiohistochemical techniques with conventional receptor binding. Their limited resolution, however, prompted us to try an immunohistochemical approach. Purified GABA/benzodiazepine receptors, prepared from bovine cerebral cortex, have been used to raise monoclonal antibodies for this purpose. Immunoreactive sites in rat brain, spinal cord and retina as well as in bovine and post-mortem human brain were found to be concentrated on neuronal cell bodies and processes in those regions known to be innervated by GABAergic neurons. Electron microscopic analysis revealed a selective staining of axosomatic and axodendritic pre- and postsynaptic contacts.     

Functional significance of the lipid-protein interface in photosynthetic membranes

The functional significance of the lipid-protein interface in photosynthetic membranes, mainly in thylakoids, is reviewed with emphasis on membrane structure and dynamics. The lipid-protein interface is identified primarily by the restricted molecular dynamics of its lipids as compared with the dynamics in the bulk lipid phase of the membrane. In a broad sense, lipid-protein interfaces comprise solvation shell lipids that are weakly associated with the hydrophobic surface of transmembrane proteins but also include lipids that are strongly and specifically bound to membrane proteins or protein assemblies. The relation between protein-associated lipids and the overall fluidity of the thylakoid membrane is discussed. Spin label electron paramagnetic resonance spectroscopy has been identified as the technique of choice to characterize the protein solvation shell in its highly dynamic nature; biochemical and direct structural methods have revealed an increasing number of protein-bound lipids. The structural and functional roles of these protein-bound lipids are mustered, but in most cases they remain to be determined. As suggested by recent data, the interaction of the non-bilayer-forming lipid, monogalactosyldyacilglycerol (MGDG), with the main light-harvesting chlorophyll a/b-binding protein complexes of photosystem-II (LHCII), the most abundant lipid and membrane protein components on earth, play multiple structural and functional roles in developing and mature thylakoid membranes. A brief outlook to future directions concludes this review.     

Structural determinants of protein folding

The last several decades have seen an explosion of knowledge in the field of structural biology. With critical advances in spectroscopic techniques in examining structures of biomacromolecules, in maturation of molecular biology techniques, as well as vast improvements in computation prowess, protein structures are now being elucidated at an unprecedented rate. In spite of all the recent advances, the protein folding puzzle remains as one of the fundamental biochemical challenges. A facet to this empiric problem is the structural determinants of protein folding. What are the driving forces that pivot a polypeptide chain to a specific conformation amongst the vast conformation space? In this review, we shall discuss some of the structural determinants to protein folding that have been identified in the recent decades.     

Iron-sulfur proteins: Recent developments in the field

Summary Iron-sulfur clusters in proteins are now recognized as among the main types of electron-transferring groups in biological systems, besides heme and flavins. Recent developments have brought forth a better understanding about the ways the protein environment modulates the potential of the cluster by placing the cluster in a more or less hydrophobic surrounding. Refinement in models, extensive studies on the kinetics of electron transfer (e.g. by measurement of the electronic spin lattice relaxation time) and the introduction of novel spectroscopic methods (EXAFS, magnetic CD and others) in the elucidation of structures in various systems are among the main developments. Other advances include EPR studies of the spatial orientation of Fe−S centers in complex membraneous systems (e.g. in mitochondria) and the recent elucidation of the nature of center X in photosystem I by M?ssbauer-spectroscopy. M?ssbauer studies have also been described on a number of Fe−S proteins (nitrogenase, aconitase, some ferredoxins, etc.) and revealed the existence of novel structures that enlarged the number of known basic units of Fe−S centers. These advances include: 1. the discovery of a novel non-heme Fe-protein (called desulforedoxin) of the rebredoxin type, 2. the elucidation of the nitrogenase Fe−S centers and the nitrogenase cofactor and 3. the discovery of a three-iron cluster in several enzymes and some ferredoxins. The latter 3-Fe cluster seems capable of being converted into a classical 4-Fe cluster under appropriate conditions, a phenomenon that plays a role in activation-deactivation of some enzymes (e.g. aconitase). It is now recognized that some iron-sulfur clusters may be involved in systems devoided of any oxydation-reduction reaction and may act as sensors of the surrounding redox potential, triggering the activation/deactivation of an enzyme (cf. e.g. aconitase).     

Benzodiazepine receptors resolved

To date, attempts to map the distribution and density of benzodiazepine receptors in the CNS have been dominated by radiohistochemical techniques with conventional receptor binding. Their limited resolution, however, prompted us to try an immunohistochemical approach. Purified GABA/benzodiazepine receptors, prepared from bovine cerebral cortex, have been used to raise monoclonal antibodies for this purpose. Immunoreactive sites in rat brain, spinal cord and retina as well as in bovine and post-mortem human brain were found to be concentrated on neuronal cell bodies and processes in those regions known to be innervated by GABAergic neurons. Electron microscopic analysis revealed a selective staining of axosomatic and axodendritic pre- and postsynaptic contacts.     

Isolation and purification of proteoglycans

Purification of a protein typically involves development of a quantitative assay to track protein integrity (e.g. enzyme activity) during subsequent isolation steps. The generalized procedure involves choosing the source of the protein, defining extraction conditions, developing bulk purification methods followed by refined, more selective methods. The purification of proteoglycans is often complicated by a) limited source quantities, b) necessity of chaotropic solvents for efficient extraction, c) their large molecular size and d) lack of defined functions to enable purity (i.e. activity, conformation) to be assessed. Because the usual goal of proteoglycan purification is physical characterization (intact molecular weight, core protein and glycosaminoglycan class and size), the problems of a suitable assay and/or native conformation are avoided. The assay for tracking proteoglycan isolation typically utilizes uronic acid content or radiolabel incorporation as a marker. Once extracted from their cellular/extracellular environment, proteoglycans can be isolated by density gradient centrifugation and/or column chromatography techniques. Recent advances in the composition of chromatographic supports have enabled the application of ion-exchange, gel permeation, hydrophobic interaction and affinity chromatography resins using efficient high-pressure liquid chromatography to proteoglycan purification.