Protein crystallography at sub-zero temperatures: lysozyme-substrate complexes in cooled mixed solvents.

To determine the feasibility of direct X-ray crystallographic structure determination of productive enzyme-substrate complexes and to ascertain the best conditions for such studies, the hydrolysis of bacterial cell walls and oligosaccharides by human leukaemic lysozyme was investigated in mixed aqueous/organic solvents and high salt solutions. Although high salt solutions modify the enzymic reaction, hydrolysis in mixed solvents appears to proceed by the same mechanism as in aqueous solution. At low temperatures the reaction is slowed progressively, and at −25 °C the enzyme-substrate complex in mixed solvents is stable indefinitely. The conformation of the enzyme is not significantly altered in these solvents, and the enzyme-substrate complex can be formed by direct addition of substrate to the enzyme at sub-zero temperatures, as required for crystallographic studies. The pH profile of the reaction in mixed solvents allows conditions of optimal binding to be selected. These studies in solution demonstrate that low-temperature protein crystallography may indeed permit the direct determination of the three-dimensional structure of enzyme-substrate complexes. They also delineate the precise conditions of pH, temperature and solvent to use in the crystallographic experiments.     

Conformational aspects of beta-trypsin interactions with substrates and pancreatic trypsin inhibitor. II. Structure of tetrahedral adducts and acylenzyme

Theoretical conformational analysis of the tetrahedral complexes of trypsin with the N-acetyl-L-lysine methyl amide, which are formed at the acylation and the deacylation stages of the catalytical act has been carried out. The lowest energy conformations are shown to be productive ones. All favorable structures of N-acetyl-L-lysyl-trypsin and N-acetyl-L-arginyl-trypsin acylenzymes have been analysed. The global conformations of both complexes are found to be very similar with the structures providing for a transition to the second tetrahedral state. Conformations of the nonbonded, tetrahedral and acyl complexes with N-acetyl-L-lysine methyl amide are compared and the differences in orientation of atomic groups participating in the catalysis are revealed. All changes of optimal structures of the complexes indispensable for the catalytical process are shown to proceed in a spontaneous way without introduction of any intramolecular strain.     

Regulation of stromal sedoheptulose 1,7-bisphosphatase activity by pH and Mg2+ concentration

A scheme is proposed for the regulation of stromal sedoheptulose 1,7-bisphosphatase activity which enlarges upon a previously elaborated mechanism (Woodrow, I.E., and Walker, D.A. (1983) Biochim. Biophys. Acta 722, 508-516). The latter involves oxidized (inactive) and reduced (active) enzyme forms. Both the free enzymes and the enzyme-substrate complexes undergo slow oxidation/reduction. This study examines the behavior of the system under pH and Mg2+ concentration regimes that are likely to occur in the chloroplast stroma. The control of enzyme activity by pH can be described in terms of each free enzyme and enzyme-substrate complex existing in protonated and nonprotonated forms. The molecular dissociation constants for each protonation reaction were calculated from kinetic data. Mg2+ concentration changes modulate these constants. Under conditions that are likely to obtain in the stroma in the dark, the model predicts that approximately 99.1% of the enzyme will be in the inactive forms. Such inactivation is important since it would prevent the reductive pentose phosphate pathway from operating in darkness.     

Hydrogen exchange-mass spectrometry: optimization of digestion conditions

The direct linkage between folded structures of proteins and their function has increased the need for high resolution structures. In addition, there is a need for analytical methods for detecting and locating changes in the folded structures of proteins under a wide variety of conditions. The rates at which hydrogens located at peptide amide linkages undergo isotopic exchange has become the basis for an important method for detecting such structural changes. When detected by mass spectrometry, hydrogen exchange can be used to study dilute solutions of large proteins and protein complexes with very high sensitivity. To locate structural changes, labeled proteins are often digested with acid proteases to form peptides whose hydrogen/deuterium levels are determined by mass spectrometry. This approach is successful only when the protein can be digested rapidly under conditions where isotope exchange is slow. This study describes how columns packed with immobilized pepsin can be used to reduce the digestion time and to provide an effective means for separating the pepsin from the isotopically labeled fragments. These columns are part of an on-line system that facilitates both rapid digestion of low concentrations of protein and concentration of the peptides.     

Free energy differences between enzyme bound states

A theory is presented that describes the free energy difference between the enzyme-substrate (ES) and enzyme-product (EP) complexes that is expected in enzymes optimized for catalytic efficiency. In such enzymes, the free energy drop between ES and EP complexes reflects a portion of the chemical potential difference between substrates and products outside the active site under physiological conditions. Qualitative and quantitative predictions of the model are discussed and compared with experimental data. The controversy over the kinetically optimal free energy profile for an enzymatic reaction operating under constraints set forward by Albery & Knowles (1976) is resolved.     

The geometry of multisite phosphorylation

Reversible protein phosphorylation on multiple sites is a key regulatory mechanism in most cellular processes. We consider here a kinase-phosphatase-substrate system with two sites, under mass-action kinetics, with no restrictions on the order of phosphorylation or dephosphorylation. We show that the concentrations of the four phosphoforms at steady state satisfy an algebraic formula—an invariant—that is independent of the other chemical species, such as free enzymes or enzyme-substrate complexes, and holds irrespective of the starting conditions and the total amounts of enzymes and substrate. Such invariants allow stringent quantitative predictions to be made without requiring any knowledge of site-specific parameter values. We introduce what we believe are novel methods from algebraic geometry—Gröbner bases, rational curves—to calculate invariants. These methods are particularly significant because they make it possible to treat parameters symbolically without having to specify their numerical values, and thereby allow us to sidestep the parameter problem. We anticipate that this approach will have much wider applications in biological modeling.     

Zooming in on disordered systems: Neutron reflection studies of proteins associated with fluid membranes

Neutron reflectometry (NR) is an emerging experimental technique for the structural characterization of proteins interacting with fluid bilayer membranes under conditions that mimic closely the cellular environment. Thus, cellular processes can be emulated in artificial systems and their molecular basis studied by adding cellular components one at a time in a well-controlled environment while the resulting structures, or structural changes in response to external cues, are monitored with neutron reflection. In recent years, sample environments, data collection strategies and data analysis were continuously refined. The combination of these improvements increases the information which can be obtained from NR to an extent that enables structural characterization of protein–membrane complexes at a length scale that exceeds the resolution of the measurement by far. Ultimately, the combination of NR with molecular dynamics (MD) simulations can be used to cross-validate the results of the two techniques and provide atomic-scale structural models. This review discusses these developments in detail and demonstrates how they provide new windows into relevant biomedical problems. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

A Combined Quantum/Classical Molecular Dynamics Study of the Catalytic Mechanism of HIV Protease

Based on available three-dimensional structures of enzyme-inhibitor complexes, the mechanism of the reaction catalysed by HIV protease is studied using molecular dynamics simulations with molecular mechanics and combined quantum-mechanics/molecular-mechanics potential energy functions. The results support the general acid/general base catalysis mechanism, with Asp25′ protonated in the enzyme-substrate complex. In the enzyme-substrate complex, the lytic water molecule binds at a position different from the positions of the hydroxyl groups in various aspartic protease-inhibitor complexes. The carboxyl groups at the active site also adopt a different orientation. However, when the lytic water molecule approaches the scissile peptide, the reaction centre changes gradually to a conformation close to that derived from X-ray diffraction studies of various enzyme-inhibitor complexes. The proton transfer processes can take place only after the lytic water molecule has approached the scissile peptide bond to a certain degree. Qualitatively, the free-energy barrier associated with the nucleophilic attack step, which takes place at physiological pH, is comparable with the acid or base-catalysed reactions of model systems. The structure of the tetrahedral intermediate resulting from the nucleophilic attack step also indicates a straightforward pathway of the next reaction step, i.e. the breaking of the C-N bond.     

Relationship between the lifetime of an enzyme-substrate complex and the properties of the molecular environment.

A theoretical discussion of the decomposition rate constants of enzyme substrate complexes is presented, based upon an enzyme model published earlier (Damjanovich &; Somogyi,1973). These rate constants are expressed by the aid of molecular parameters characteristic for the enzyme-substrate complexes and the molecules in the surrounding liquid phase.Both the exponential and pre-exponential factors of the expressions describing the composition rate constants contain parameters depending on the mass distribution of the reaction mixture in a specific way which is characteristic for the enzyme-substrate complex. The findings suggest a new kind of the enzyme regulation generated by the surrounding medium.     

Floral abnormalities in field grown “Tigrina” plants from xHaynaldoticum sardoum Meletti et Onnis (Gramineae)

A ‘tigrina’ mutant stock of xHaynaldoticum sardoum Meletti et Onnis grown under field conditions produced high frequency spikes with abnormal stamens and pistils. The abnormalities occurred mostly in the stamens, which frequently changed to either pseudoovaries, or leaf-shaped structures or, in extreme cases, did not form at all. The developmental abnormalities of the stamens resulted in male sterility in the whole or in part of the spike. It is proposed that the “tigrina” status changes the physiology of the plant (e.g. photosynthetic activity) in such a way that it responds with floral abnormalities brought about by environmental conditions which permit normal flower development in control (non-mutant) plants.     

Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.     

Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition

Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes.     

NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms

NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.     

The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication.

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.     

Where is the glycolytic complex? A critical evaluation of present data from muscle tissue

Associations between glycolytic enzymes and subcellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzyme binding to subcellular structural components is believed to regulate enzyme activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that enzyme binding to cellular structures is not involved in the control of glycolysis. (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strength and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist. (ii) In many cases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity. (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negligible.     

Easy DNA Modeling and More with GraphiteLifeExplorer

The GraphiteLifeExplorer tool enables biologists to reconstruct 3D cellular complexes built from proteins and DNA molecules. Models of DNA molecules can be drawn in an intuitive way and assembled to proteins or others globular structures. Real time navigation and immersion offer a unique view to the reconstructed biological machinery.     

Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration

Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV‐1 that target integrase recognize the nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV‐1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance. To date, prototype foamy virus (PFV) is the only retrovirus for which such structures have been determined. We show that PFV strand transfer complexes (STC) can be assembled on product DNA without going through the normal forward reaction pathway. The finding that a retroviral STC can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in HIV‐1 DNA integration.     

Evidence for the association of core histones with poly(U) to nucleosome-like complexes

As was shown in a recent paper [Klump, H. & Hütig, H. (1980) Ber. Bunsenges. Phys. Chem. 84, 250-253] by one of the authors (H.K.), nucleosome-like histone complexes can be formed on ribopolynucleotides as well as on deoxyribopolynucleotides. The conditions used for complex formation have to be such that single strands and triple strands as secondary structures of the polynucleotides employed are excluded under all ionic-strength conditions. There is clear evidence however for ordered poly(U)/histone complexes. In this paper we further characterize these poly(U) complexes and present electron micrographs of both DNA histone complexes and poly(U) histone complexes to show their close resemblance.