Cooperation of the prolyl isomerase and chaperone activities of the protein folding catalyst SlyD

The SlyD (sensitive to lysis D) protein of Escherichia coli is a folding enzyme with a chaperone domain and a prolyl isomerase domain of the FK506 binding protein type. Here we investigated how the two domains and their interplay are optimized for function in protein folding. Unfolded protein molecules initially form a highly dynamic complex with the chaperone domain of SlyD, and they are then transferred to the prolyl isomerase domain. The turnover number of the prolyl isomerase site is very high and guarantees that, after transfer, prolyl peptide bonds in substrate proteins are isomerized very rapidly. The Michaelis constant of catalyzed folding reflects the substrate affinity of the chaperone domain, and the turnover number is presumably determined by the rate of productive substrate transfer from the chaperone to the prolyl isomerase site and by the intrinsic propensity of the refolding protein chain to leave the active site with the native prolyl isomer. The efficiency of substrate transfer is high because dissociation from the chaperone site is very fast and because the two sites are close to each other. Protein molecules that left the prolyl isomerase site with an incorrect prolyl isomer can rapidly be re-bound by the chaperone domain because the association rate is very high as well.     

Enzymatic reactions in small spatial volumes: comment on a model of Hess and Mikhailov

Recently Hess and Mikhailov pointed out that in small subcellular compartments diffusion is so fast that mixing is instantaneous on the time scale of many enzymatic reactions. This opens the possibility for synchronizing individual reaction events. To illustrate this fact they discuss as example an irreversible enzymatic reaction with allosteric product activation. Under appropriate conditions their model shows coherent spiking in the number of product molecules, caused by the strong correlation between reaction events. In this model only substrate binding is an indeterministic process, all other subsequent transitions between different enzyme states being deterministic, contrary to real processes. The purpose of the present paper was to investigate this interesting phenomenon by means of a more realistic modification of the original model, with only probabilistic transitions. In an attempt to obtain spiking, which was not observed under these conditions, the model was extended to make a clear distinction between allosteric high and low affinity substrate binding, in contrast to the original model using a product dependent mean binding probability. However no periodic signal was detectable in the indeterministic version of the Hess Mikhailov model or the extended version, either by means of direct visualization or on autocorrelation or Fourier analysis. Reasons why spiking is not observed in indeterministic enzyme models are discussed.     

A mechanism for multiphasic uptake of solutes in plants

A molecular mechanism is proposed for unitary multiphasic uptake in which the carrier has two binding sites for the substrate. The first site binds n-1 molecules of substrate, and then one additional substrate molecule can become bound at the second binding site. Only this last molecule is transported in the operation of the carrier molecule. In the free state, the carrier can be activated to successive states with increasing affinities for the substrate in the two binding sites. The mechanism is resolved for the steady state conditions, obtaining a simple uptake rate equation, which fits the experimental data. Methods for determining the parameters of the equation are presented. Evidence other than kinetics is discussed for the mechanism. The mechanism also provides a physiological interpretation for multiphasic uptake: the active transport mechanisms (energy-requiring mechanisms) are prevented from operating at high substrate concentrations, thus preventing a waste of energy by the cells.     

Substrate Binding Specifically Modulates Domain Arrangements in Adenylate Kinase

The enzyme adenylate kinase (ADK) features two substrate binding domains that undergo large-scale motions during catalysis. In the apo state, the enzyme preferentially adopts a globally open state with accessible binding sites. Binding of two substrate molecules (AMP + ATP or ADP + ADP) results in a closed domain conformation, allowing efficient phosphoryl-transfer catalysis. We employed molecular dynamics simulations to systematically investigate how the individual domain motions are modulated by the binding of substrates. Two-dimensional free-energy landscapes were calculated along the opening of the two flexible lid domains for apo and holo ADK as well as for all single natural substrates bound to one of the two binding sites of ADK. The simulations reveal a strong dependence of the conformational ensembles on type and binding position of the bound substrates and a nonsymmetric behavior of the lid domains. Altogether, the ensembles suggest that, upon initial substrate binding to the corresponding lid site, the opposing lid is maintained open and accessible for subsequent substrate binding. In contrast, ATP binding to the AMP-lid induces global domain closing, preventing further substrate binding to the ATP-lid site. This might constitute a mechanism by which the enzyme avoids the formation of a stable but enzymatically unproductive state.     

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.     

Modeling Facilitative Sugar Transporters: Transitions Between Single and Double Ligand Occupancy of Multiconformational Channel Models Explain Anomalous Kinetics

The four-state simple carrier model (SCM) is employed to describe ligand translocation by diverse passive membrane transporters. However, its application to systems like facilitative sugar transporters (GLUTs) is controversial: unidirectional fluxes under zero-trans and equilibrium-exchange experimental conditions fit a SCM, but flux data from infinite-cis and infinite-trans experiments appear not to fit the same SCM. More complex kinetic models have been proposed to explain this ``anomalous' behavior of GLUTs, but none of them accounts for all the experimental findings. We propose an alternative model in which GLUTs are channels subject to conformational transitions, and further assume that the results from zero-trans and equilibrium-exchange experiments as well as trans-effects corresponds to a single-occupancy channel regime, whereas the results from the infinite-cis and infinite-trans experiments correspond to a regime including higher channel occupancies. We test the plausibility of this hypothesis by studying a kinetic model of a two-site channel with two conformational states. In each state, the channel can bind the ligand from only one of the compartments. Under single-occupancy, for conditions corresponding to zero-trans and equilibrium-exchange experiments, the model behaves as a SCM capable of exhibiting trans-stimulations. For a regime including higher degrees of occupancy and infinite-cis and infinite-trans conditions, the same channel model can exhibit a behavior qualitatively similar to a SCM, albeit with kinetic parameters different from those for the single-occupancy regime. Numerical results obtained with our model are consistent with available experimental data on facilitative glucose transport across erythrocyte membranes. Hence, if GLUTs are multiconformational channels, their particular kinetic properties can result from transitions between single and double channel occupancies. Received: 12 April 1995/Revised: 28 August 1995     

Displacement of a DNA binding protein by Dda helicase

Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase.     

Water permeation through the sodium-dependent galactose cotransporter vSGLT

It is well accepted that cotransporters facilitate water movement by two independent mechanisms: osmotic flow through a water channel in the protein and flow driven by ion/substrate cotransport. However, the molecular mechanism of transport-linked water flow is controversial. Some researchers believe that it occurs via cotransport, in which water is pumped along with the transported cargo, while others believe that flow is osmotic in response to an increase in intracellular osmolarity. In this letter, we report the results of a 200-ns molecular dynamics simulation of the sodium-dependent galactose cotransporter vSGLT. Our simulation shows that a significant number of water molecules cross the protein through the sugar-binding site in the presence as well as the absence of galactose, and 70-80 water molecules accompany galactose as it moves from the binding site into the intracellular space. During this event, the majority of water molecules in the pathway are unable to diffuse around the galactose, resulting in water in the inner half of the transporter being pushed into the intracellular space and replaced by extracellular water. Thus, our simulation supports the notion that cotransporters act as both passive water channels and active water pumps with the transported substrate acting as a piston to rectify the motion of water.     

Quantitative relationships of site to site interaction in Escherichia coli D-3-phosphoglycerate dehydrogenase revealed by asymmetric hybrid tetramers

A set of asymmetric hybrid tetramers of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) have been made by gene co-expression and KSCN-induced dimer exchange. These tetramers contain varied numbers of active sites and effector binding sites arranged in different orientations within the tetramer. They reveal that PGDH displays half-of-the-sites activity with respect to its active sites and that the two sites that are active at any particular time lie in subunits on either side of the nucleotide binding domain interface. Half-of-the-sites functionality is also observed for the effector even though all four sites eventually bind effector. That is, only two effector sites need to be occupied for maximum inhibition. Binding of the last two effector molecules does not contribute functionally to inhibition of activity. Furthermore, positive cooperativity of inhibition of activity by the effector is completely dependent on the positive cooperativity of binding of the effector. Binding of the first effector molecule produces a conformational change that essentially completely inhibits the active site within the subunit to which it binds and produces an approximate 33% inhibition of the active site in the subunit to which it is not bound. Binding of the second effector at the opposite regulatory domain interface completes the inhibition of activity. This simple relationship defines the positional and quantitative influence of effector ligand binding on activity and can be used to predict the maximum level of inhibition of individual hybrid tetramers. In addition, the site-specific quantitative relationship of effector binding to individual active sites can be used to model the inhibition profile with relatively good agreement. These simple rules for the site to site interaction in PGDH provide significant new insight into the mechanism of allostery of this enzyme.     

Basic residues are important for Ca2+/calmodulin binding and activation but not autoinhibition of rabbit skeletal muscle myosin light chain kinase

Several allosterically modulated protein kinases have been shown to be regulated by an autoinhibitory domain located within the kinase molecules. The inhibitory domain has been proposed to act as a "pseudosubstrate" inhibitor binding to the substrate binding site of the kinase, thereby blocking the binding of the enzyme's true substrate. In this report, site-directed mutagenesis has been used to further investigate the mechanism of activation of the inhibitory domain of rabbit skeletal muscle myosin light chain kinase. Basic residues within the pseudosubstrate domain (572-573, 577-579, 580-581), which are analogous to the important substrate determinants of the myosin light chain, were found not to be required in order to maintain the kinase in an inhibited state. Two groups of these residues (577-579 and 581-582) were, however, found to be important for high affinity calmodulin binding to the kinase. These data suggest that the autoinhibitory domain of myosin light chain kinase may not function by directly mimicking the light chain substrate.     

Cleavage mediated by the P15 domain of bacterial RNase P RNA

Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3'-end of tRNA precursors resulting in the formation of the RCCA-RNase P RNA interaction (interacting residues underlined) in the bacterial RPR-substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg(2+) at the cleavage site. Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for 'RNA-mediated' cleavage of RNA.     

Evidence for induced fit in bacterial RNase P RNA-mediated cleavage

RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg(2+) and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg(2+)) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.     

A crystallographic study of the glutathione binding site of glutathione reductase at 0.3-nm resolution

The binding of glutathione, some related molecules and two redox compounds to crystals of glutathione reductase has been investigated by X-ray crystallography at 0.3-nm resolution. Models for several bound ligands have been built and subjected to crystallographic refinement. The results clearly show the residues involved in glutathione binding as well as the geometry of the disulfide exchange. Glutathione-I is bound in a V-shaped conformation, while glutathione-II is extended. The zwitterionic glutamyl end of glutathione-II appears to be the most tightly bound part of the substrate. All glutathione conjugates and derivatives studied show binding dominated by the interactions at this site. In the reduced enzyme, glutathione-I forms a mixed disulfide intermediate with Cys58. Other structural changes are observed on reduction of the enzyme, and it is demonstrated that the carboxamidomethylated enzyme is a good model for the reduced species. Lipoate, a weak substrate of the enzyme, assumes a defined binding site where its disulfide is available for being attacked by Cys58-S gamma. A second region with affinity for a number of compounds has been found in a large cavity at the dimer interface of the enzyme. No functional role of this site is known.     

Mutations affecting the activity of urokinase-type plasminogen activator.

Mutagenesis throughout the single-chain urokinase-type plasminogen activator (scu-PA) cDNA molecule, followed by expression of the mutant genes and secretion of the resulting mutant proteins from yeast, has been used to determine the amino acid residues important for activity of scu-PA molecules. Twelve out of 13 colonies secreting variant scu-PA molecules with decreased ability to form a zone of fibrinolysis had mutant genes with a single codon alteration in the serine protease encoding domain (B-chain). Many of these changes are of highly conserved residues in the serine proteases and are consequently of considerable interest. A model three-dimensional structure of the protease domain of urokinase was used to explain the basis for the effects of these down mutations. The model showed that the strongest down mutations result from either interference of the mutated side chain with substrate binding at the active site or the introduction of bulky or charged groups at structurally sensitive internal positions in the molecule. Attempts to find second site revertants of five down mutants, altered either at the plasmin activation site or near the serine at the active site, only resulted in same-site revertants, with the original or closely related amino acids restored.     

Domain motion and interdomain hot spots in a multidomain enzyme

The aim of this article is to analyze conformational changes by comparing 10 different structures of Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase (PMM/PGM), a four‐domain enzyme in which both substrate binding and catalysis require substantial movement of the C‐terminal domain. We focus on changes in interdomain and active site crevices using a method called computational solvent mapping rather than superimposing the structures. The method places molecular probes (i.e., small organic molecules containing various functional groups) around the protein to find hot spots. One of the most important hot spots is in the active site, consistent with the ability of the enzyme to bind both glucose and mannose phosphosugar substrates. The protein has eight additional hot spots at domain‐domain interfaces and hinge regions. The locations and nature of six of these hot spots vary between the open, half‐open, and closed conformers of the enzyme, in good agreement with the ligand‐induced conformational changes. In the closed structures the number of probe clusters at the hinge region significantly depends on the position of the phosphorylated oxygen in the substrate (e.g., glucose 1‐phosphate versus glucose 6‐phosphate), but the protein remains almost unchanged in terms of the overall RMSD, indicating that computational solvent mapping is a more sensitive approach to detect changes in binding sites and interdomain crevices. Focusing on multidomain proteins we show that the subresolution conformational differences revealed by the mapping are in fact significant, and present a general statistical method of analysis to determine the significance of rigid body domain movements in X‐ray structures.     

First structural evidence for the mode of diffusion of aromatic ligands and ligand-induced closure of the hydrophobic channel in heme peroxidases

The mode of binding of aromatic ligands in the substrate binding site on the distal heme side in heme peroxidases is well understood. However, the mode of diffusion through the extended hydrophobic channel and the regulatory role of the channel are not yet clear. To provide answers to these questions, the crystal structure of the complex of lactoperoxidase and 3-amino-1,2,4-triazole (amitrole) has been determined, which revealed the presence of two ligand molecules, one in the substrate binding site and the second in the hydrophobic channel. The binding of ligand in the channel induced a remarkable conformational change in the side chain of Phe254, which flips from its original distant position to interact with the trapped ligand in the hydrophobic channel. As a result, the channel is completely blocked so that no ligand can diffuse through it to the substrate binding site. Another amitrole molecule is bound to lactoperoxidase in the substrate binding site by replacing three water molecules, including the crucial iron-bound water molecule, W1. In this arrangement, the amino nitrogen atom of amitrole occupies the position of W1 and interacts directly with ferric iron. As a consequence, it prevents the binding of H₂O₂ to heme iron. Thus, the interactions of amitrole with lactoperoxidase obstruct both the passage of ligands through the hydrophobic channel as well as the binding of H₂O₂. This explains the amitrole toxicity. From binding studies, the dissociation constant (K _d) for amitrole with lactoperoxidase was found to be approximately 5.5 × 10⁻⁷ M, indicating high affinity.     

Crystal structure of a transcarbamylase-like protein from the anaerobic bacterium Bacteroides fragilis at 2.0 A resolution

A transcarbamylase-like protein essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis has been purified and crystallized in space group P4(3)2(1)2 (a=b=153.4 A, c=94.8 A). The structure was solved using a single isomorphous replacement with anomalous scattering (SIRAS) and was refined at 2.0 A resolution to an R-factor of 20.6% (R-free=25.2%). The molecular model is trimeric and comprises 960 amino acid residues, two phosphate groups and 422 water molecules. The monomer has the consensus transcarbamylase fold with two structural domains linked by two long interdomain helices: the putative carbamoyl phosphate-binding domain and a binding domain for the second substrate. Each domain has a central parallel beta-sheet surrounded by alpha-helices and loops with alpha/beta topology. The putative carbamoyl phosphate-binding site is similar to those in ornithine transcarbamylases (OTCases) and aspartate transcarbamylases (ATCases); however, the second substrate-binding site is strikingly different. This site has several insertions and deletions, and residues critical to substrate binding and catalysis in other known transcarbamylases are not conserved. The three-dimensional structure and the fact that this protein is essential for arginine biosynthesis suggest strongly that it is a new member of the transcarbamylase family. A similar protein has been found in Xylella fastidiosa, a bacterium that infects grapes, citrus and other plants.