Essential dynamics of the cellular retinol-binding protein evidence for ligand-induced conformational changes

The cellular retinol-binding protein (CRBP) is an intracellularretinol carrier protein belonging to a family of hydrophobicligand–binding proteins. It transports retinol to specificlocations in the cell where, for instance, it is esterifiedfor storage. Recently solved crystallographic structures ofCRBP homologues with and without bound ligand do not provideevidence for a ligand–induced conformational change. However,it has been shown that there is a difference in binding of holo–CRBPand apo–CRBP to lecithin–retinol acyltransferase.Moreover, proteolysis of holo–CRBP and apo–CRBPyields different products, indicating a difference in structureor dynamics between the two forms. Here, we present the resultsof molecular dynamics simulations of holo–CRBP and apo–CRBP.The simulations show a significant difference in conformation,in agreement with experimental results. The essential dynamicsmethod was used to study differences in dynamics between theapo and holo forms of CRBP, and showed inhibition of essentialmotions upon ligand binding. It also revealed large correlatedmotions of retinol with regions of the protein, pointing toa possible retinol entry/exit site.     

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease

Using genetic techniques the contribution of surface loops tothe thermal stability of Bacillus subtUis neutral protease (NPsub)wasstudied. Mutations were designed to make the surface of NP-submore similar to the surface of more thermostable neutral proteasessuch as thermolysin (TLN). The mutations included the replacementof an irregular loop by a shorter variant and the introductionof a ten–residue (3– hairpin. In general, thesedrastic mutations had little effect on the production and activityof NP–sub, indicating the feasibility of major structuralrearrangements at the surface of proteins. In the most stablemutant, exhibiting an increase in thermal stability of 1.1°C, 10% of the surface of NP–sub was modified. Several NP–subvariants carrying multiple mutations were constructed. Non–additiveeffects on thermal stability were observed, which were interpretedon the basis of a model for thermal inactivation, that emphasizesthe importance of local unfolding processes for thermal stability     

The mechanism of sequence non-specific DNA binding of HMG1/2-box B in HMG1 with DNA

The DNA binding mechanism of box B in HMG1, a member of thesequence non-specific DNA binding HMG1/2-box family of proteins,has been examined by both mutation analyses and molecular modelingtechniques. Substitution of the residue 102F, which is characteristicallyexposed to solvent, with a small hydrophobic amino acid affectedits DNA binding activity. However, no additional effect wasobserved by the further mutation of flanking 101F. Moleculardynamics simulation and modeling studies revealed that 102Fintercalates into DNA base-pairs, being supported by the flanking101F. The mutants with a small hydrophobic residue at position102 tolerated the substitution for 101F because the side chainat position 102 is too short to intercalate. Thus the intercalationof 102F and the positive effect of the flanking 101F residueare important for the sequence non-specific DNA binding of theHMG1/2-box. The conserved basic residues of 95K, 96R and 109Rwere also examined for their roles in DNA binding. These residuesinteracted with DNA mainly by electrostatic interaction andmaintained the location of the box on the DNA, which prescribedthe intercalation of 102F. The DNA intercalation by HMG1 consistsof an ingenious mechanism which brings DNA conformational changesnecessary for biological functions.     

Exploring the conformational diversity of loops on conserved frameworks

Loops are structurally variable regions, but the secondary structuralelements bracing loops are often conserved. Motifs with similarsecondary structures exist in the same and different proteinfamilies. In this study, we made an all-PDB-based analysis andproduced 495 motif families accessible from the Internet. Everymotif family contains some variable loops spanning a commonframework (a pair of secondary structures). The diversity ofloops and the convergence of frameworks were examined. In addition,we also identified 119 loops with conformational changes indifferent PDB files. These materials can give some directionsfor functional loop design and flexible docking.     

Enzyme-catalyzed dehalogenation of pentachloroethane: why F87W-cytochrome P450cam is faster than wild type

Under anaerobic conditions, cytochromes P450 can reductivelydehalogenate heavily halogenated hydrocarbons, such as one-and two-carbon organic solvents. This catalytic capacity hasdrawn attention to the potential use of engineered forms ofP450s in the remediation of contaminated deep subsurface ecosystems.Loida (1994, PhD Thesis, University of Illinois at Urbana-Champaign,IL) and S.G.Sligar (personal communication) have observedrecentlythat an active-site variant of cytochrome P450cam (F87W) dechlorinatespentachloroethane approximately three times faster than thewild-type enzyme. Molecular dynamics simulations have revealedthat the mutant enzyme binding pocket remains smaller, and thatpentachloroethane assumes configurations closer to the heme-Fein the F87W mutant twice as often as in the wild-type enzyme.This result is consistent with a collisional model of dehalogenation,which agrees with experimental observations [Li and Wackett(1993) Biochemistry, 32, 9355–9361] that solutions containingwild-type P450cam dehalogenate pentachloroethane 100 times fasterthan those containing free heme. The simulations suggest thatit is unlikely that Trp87 significantly stabilizes the developingnegative charge on the substrate during carbon-halogen bondreduction. The design of improved microbiai enzymes that incorporateboth steric and electronic effects continues for use in remediatinghalogenated contaminants in situ     

Molecular modeling of coiled-coil {alpha}-tropomyosin: analysis of staggered and in register helix--helix interactions

In register and staggered models of tropomyosin coiled-coilwere built from X-ray C coordinates and refined via moleculardynamics. The two models show similar structural features withthe X-ray structure of GCN4 leucine zipper. Empirical energeticmethods used to compare the in register and staggered modelsindicate that both are equally probable. The two models havesimilar profiles of solvation free energy of folding for residuesat positions a and d of the repeating heptad, indicating thatresidues at these positions are as well buried in an in registerstructure as in a staggered one. Neither the in register northe 14 residues staggered structure can be ruled out based onhydrophobic or e–g' (g–e') electrostatic interactionswhich are not able to distinguish between the two models andare therefore not selective. However, the eg–b'c' electrostaticinteractions, although smaller in magnitude, are in favor ofthe in register model. Furthermore, analysis of hydrophobicand electrostatic interactions along the tropomyosin sequenceshows that bulky residues in positions a and d prevent the formationof inter-chain salt bridges.     

Different disease-causing mutations in transthyretin trigger the same conformational conversion

Transthyretin (TTR)-containing amyloid fibrils are deposited in cardiac tissue as a natural consequence of aging. A large number of inherited mutations lead to amyloid diseases by accelerating TTR deposition in other organs. Amyloid formation is preceded by a disruption of the quaternary structure of TTR and conformational changes in the monomer. To study conformational changes preceding the formation of amyloid, we performed molecular dynamics simulations of the wild-type monomer, amyloidogenic variants (V30M, L55P, V122I) and a protective variant (T119M) at neutral and low pH. At low pH, the D strand dissociated from the beta-sheet to expose the A strand, consistent with experimental studies. In amyloidogenic variants and in the wild-type at low pH, there was a conformational change in the beta-sheets into alpha-sheet via peptide bond flips that was not observed at neutral pH in the wild-type monomer. The same residues participated in conversion in each amyloidogenic variant simulation, originating in the G strand between residues 106 and 109, with accelerated conversion at low pH. The T119M protective variant changed the local conformation of the H strand and suppressed the conversion observed in amyloidogenic variants.     

Destabilizing interactions between the partners of a bifunctional fusion protein

Hybrid MalE–GVP is a bifunctional protein in vitro sinceit binds maltose as protein MalE of Escherichia coli and sinceit is dimeric and specifically binds single-stranded DNA asprotein GVP of phage M13. The oxidation rate of a unique cysteineresidue was used to compare the stabilities of GVP in its freeand hybrid forms, under conditions where MalE was either foldedor unfolded by a denaturing agent. The results showed that boththe covalent link and tertiary non-covalent interactions betweenMalE and GVP destabilized GVP in MalE–GVP. To test whetherGVP had identical structures in its free and hybrid forms, mutationswere used as local conformational probes. The effects of thesemutations on the capabilities of MalE–GVP to dimerizeand to bind single-stranded DNA were assayed in vitro. Theywere compatible with the effects of the same mutations on theglobal activity of free GVP in vivo and with the effects thatcould be predicted from the known data on free GVP, in particularits crystal structure. Thus, one partner of a hybrid proteincan be destabilized by the other partner while maintaining itsstructural and functional characteristics.     

The role of the flap residue, threonine 77, in the activation and catalytic activity of pepsin A

Flexible loops, often referred to as flaps, have been shownto play a role in catalytic mechanisms of different enzymes.Flaps at the active site regions have been observed in the crystalstructures of aspartic proteinases and their residues implicatedin the catalytic processes. This research investigated the roleof the flap residue, threonine 77, in the activation of pepsinogenand the catalytic mechanism of pepsin. Three mutants, T77S,T77V and T77G, were constructed. Differences in amino acid polarityand hydrogen bonding potential were shown to have an influenceon the activation and catalytic processes. T77S activated atthe same rate and had similar catalytic parameters as the wild-typepepsin. The activation rates of T77V and T77G were slower andtheir catalytic efficiencies lower than the wild-type. The resultsdemonstrated that the threonine 77 polar side chain played arole in a proteolysis. The contribution of the side chain tozymogen activation was associated with the proteolytic cleavageof the prosegment. It was postulated that the hydroxyl groupat position 77 provided an essential hydrogen bond that contributedto proper substrate alignment and, indirectly, to a catalyticallyfavorable geometry of the transition state.     

Conservation and specialization in PAS domain dynamics

The PAS (Per-ARNT-Sim) superfamily is presented as a well-suited study case to demonstrate how comparison of functional motions among distant homologous proteins with conserved fold characteristics may give insight into their functional specialization. Based on the importance of structural flexibility of the receptive structures in anticipating the signal-induced conformational changes of these sensory systems, the dynamics of these structures were analysed. Molecular dynamics was proved to be an effective method to obtain a reliable picture of the dynamics of the crystal structures of HERG, phy3, PYP and FixL, provided that an extensive conformational space sampling is performed. Other reliable sources of dynamic information were the ensembles of NMR structures of hPASK, HIF-2alpha and PYP. Essential dynamics analysis was successfully employed to extract the relevant information from the sampled conformational spaces. Comparison of motion patterns in the essential subspaces, based on the structural alignment, allowed identification of the specialized region in each domain. This appears to be evolved in the superfamily by following a specific trend, that also suggests the presence of a limited number of general solutions adopted by the PAS domains to sense external signals. These findings may give insight into unknown mechanisms of PAS domains and guide further experimental studies.     

Molecular modelling of UH-301 and 5-HTla receptor interactions

A three-dimensional model of the human 5-HT_1a receptor was constructedby molecular modelling, and the molecular and electronic structuresof (R)- and (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin(UH-301) and of (R)- and (S)-8-hydroxy-2-(dipropylamino)tetralin(8-OH-DPAT) were examined by molecular mechanics and quantummechanics calculations and molecular dynamics simulations. Thereceptor model has seven transmembrane helices (TMHs), organizedaccording to a projection map of visual rhodopsin, and includesall loops between helices and the N- and C-terminal parts. Interactionsof UH-301 and 8-OH-DPAT with the 5-HT_1a receptor were examinedby molecular dynamics simulations and energy minimization ofreceptor–ligand complexes. 8-OH-DPAT had lower electrostaticpotentials around the hydroxyl group and stronger hydrogen bondingto the receptor model than had UH-301. The simulations indicatedthat the 5-HT_1a receptor agonists, (R)- and (S)-8-OH-DPAT and(R)-UH-301, interacted with the receptor at a site closer toAsp82 in TMH2 than did (S)-UH-301, which is a 5-HT,_1a receptorantagonist. Simulations of receptor–ligand complexes indicatedthat Asp82, Asp116, Ser199, Thr200 and De385 are essential forbinding of both agonist and antagonist to the receptor.     

Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases

The Streptomyces subtilisin inhibitor (SSI) is a proteinaceousprotease inhibitor which inhibits serine proteases by forminga stable Michaelis complex. The flexible loop region (Thr64–Val69)is a very flexible region in an SSI molecule and its importancein interactions with proteases has been suggested, since conformationalchange of this loop was found to occur for the smooth bindingof SSI with various proteases. In this study, mutated SSIs lackingone or two residues in this region were generated and the effectsof deletions on the interaction with proteases were investigated.Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73)both known to be trypsin inhibitors, to examine the effectsof deletion on interactions with subtilisin BPN' or trypsin.The deletion of one residue (Gly66) caused increased inhibitoryactivity toward trypsin, indicating the protruding flexibleloop hinders binding with trypsin. Reduction of such hindranceby one-residue shortening in this loop is shown to be effectivefor the interaction of SSI(Lys73) with trypsin. In contrast,one-residue shortening had virtually no effect on inhibitiontoward subtilisin BPN'. Differences in the subsite structuresof these proteases may have been the reason for this contrast.The deletion of two residues (Thr64 and Gly66) in this regionconverted SSI into a temporary inhibitor. Structural analysisof the degradation intermediate showed that the peptide bondat the reactive site of doubly deleted SSI was cleaved by subtilisinBPN' after its binding with protease. Thus, the irreversibilityof the cleaved peptide bond at the reactive site of mutatedSSI in the complex with protease may possibly be the cause forits temporary inhibition. Irregular conformation around thereactive site caused by the deletion of two residues in theflexible loop would convert SSI into a temporary inhibitor.Thus, moderate flexibility in the flexible loop region may possiblybe a structural requirement for SSI to function.     

Can the stability of protein mutants be predicted by free energy calculations?

The use of free energy simulation techniques in the study ofprotein stability is critically evaluated. Results from twosimulations of the thermostability mutation Asn218 to Ser218in Subtilisin are presented. It is shown that components ofthe free energy change can be highly sensitive to the computationaldetails of the simulation leading to the conclusion that freeenergy calculations cannot currently be used to reliably predictprotein stability. The different factors that undermine thereliability are discussed.     

Substrate mobility in thiocamphor-bound cytochrome P450cam: an explanation of the conflict between the observed product profile and the X-ray structure

Thiocamphor is an unusual substrate for P450_cam in that in theX-ray structure it binds in the active site pocket in two distinctorientations and neither of these orientations are consistentwith the 5-alcohol being the primary product. Other camphoranalogs such as norcamphor or camphane bind in a single orientationconsistent with the 5-alcohol being a major product. We presentan analysis of four 175 ps molecular dynamics trajectories ofthiocamphor-bound cytochrome P450_cam. The first two trajectorieswere calculated for cytochrome P450_cam with thiocamphor boundin both its major and minor crystallographic orientations. Inthe second set of simulations, a single oxygen atom was addedas a distal ligand to the heme group in order to model the putativeferryl oxygen reaction intermediate. Trajectories were againcalculated starting with thiocamphor in its major and minororientations. While the protein dynamics were quite similarin all four trajectories, the substrate showed distinctly differentmotions in each of the trajectories. In particular, the preferredsubstrate orientations were very different in the presence ofthe ferryl oxygen than in the absence of that oxygen. The preferredorientations in the absence of the distal oxygen were consistentwith the 3-akohol being the major product, while the preferredorientations in the presence of the distal oxygen were consistentwith the 5-alcohol being a major product. These simulationsoffer an explanation for the inconsistency between the X-raydata and the product profile.     

Simulation of a complex protein structural change: the T {leftrightarrow} R transition in the insulin hexamer

The T R transition in the insulin hexamer is an outstandingmodel for protein structural changes in terms of its extentand complexity: the limiting structures T₆, T₃R₃ and R₆ havebeen defined by X-ray crystallography. The transition occurscooperatively within trimers. It involves displacements of >30Å and a secondary structural rearrangement of 15% of thepeptide chain between extended and helical conformations. Experimentaldata for the transition are plentiful. Theoretical methods tosimulate pathways without constraints would never succeed withsuch substantial transitions. We have developed two approaches,targeted energy minimization (TEM) and targeted molecular dynamics(TMD). Previously successful in simulating the T R transitionof the insulin monomer, these procedures are also shown hereto be effective in the hexamer. With TMD, more conformationalspace is explored and pathways are found at 500 kJ/mol lowerenergy than with TEM. Because the atoms have to meet distanceconstraints in sum rather than individually, a high degree ofconformational freedom and independence. is implied. T₆ T₃R₃and T₃R₃ T₆ pathways do not coincide because the transformationis directed. One subunit enters a dead end pathway in one directionof the TMD simulation, which shows that constraint and freedomare critically balanced. The ensemble of productive pathwaysrepresents a plausible corridor for the transition. A videodisplay of the transformations is available     

Molecular dynamics simulation of trp-aporepressor in a solvent

Molecular dynamics simulations of Escherichia coli trp-aporepressorwere carried out in the absence and presence of explicit watermolecules. The vacuum simulations resulted in significant deformationof the initial X-ray structure. A solvated simulation with anonbonded cut-off radius of 9 Å gave a better result,and the most satisfactory result was obtained when electrostaticinteractions within a cut-off radius of 18 Å were consideredby a twin-range method. The trajectory from the last simulationwas used to analyze the dynamical properties of the aporepressor.The root-mean-square fluctuations of the residues showed therigidity of the central core and the flexibility of the DNA-bindingsites, consistent with the X-ray temperature factors. The dynamicalcross-correlation map indicated a significant negative correlationbetewen the central core and the two DNA-binding sites, andthus reproduced the three-domain format (a central core andtwo DNA-binding heads) from a dynamical point of view. The coreregion showed weak, but many, intra- and inter-molecular correlations,while the helix-turn-helix DNA-binding motifs were free fromcorrelations with other regions.     

On the stability and plastic properties of the interior L3 loop in R.capsulatus porin. A molecular dynamics study

Structural properties of Rhodobacter capsulatus porin are studiedby molecular dynamics simulation using the GROMOS force field.Unconstrained simulations of the trimer and monome r show thetrimer to be more stable than the isolated monomer. Simulationsof the L3 loop insi de the pore are used to assess its stabilityand plastic properties. Simulated annealing shows that the conformationalspace available to the L3 loop inside the pore is very large.Simulations at different temperatures show that the energy hypersurfacearound the open state is complex and flat. These studies alsoindicate four zones that are more flexible than the rest ofthe loop. Two of these are stabilized by the addition of thedetergent molecule present in the X-ray structure. It is possiblethat the two remaining flexible zones, situated in the halfof the loop facing the extracellular end of the porin molecule,residues Asp93–Gly98 and Arg110–Leu111, are involvedin a mechanism for opening and closing of the pore.     

Estimation of binding free energies for HIV proteinase inhibitors by molecular dynamics simulations

Absolute binding free energies for three inhibitors of HIV–1proteinase were estimated from molecular dynamics simulationsby a recently reported linear approximation procedure. The resultswere in fairly good agreement with experimental binding data.Two of the inhibitors were very similar and, for comparison,their relative free energies of binding were also calculatedby free energy perturbation methods, giving virtually the sameresult Effects of cutoff radii and charge states of the proteinmodel were examined. The effects of pH on binding of one ofthe inhibitors were predicted.     

Properties of N-terminal tails in G-protein coupled receptors: a statistical study

The extramembraneous segments in a large collection of G-proteincoupled receptors (GPCRs) have been analysed in terms of aminoacid composition and length. It is shown that this family ofmulti-spanning integral membrane proteins conforms well to the‘positive inside’ rule. Further, the extracellularN-terminal tails of GPCRs lacking a cleavable signal peptideare shown to be considerably shorter and to have a reduced contentof positively charged amino acids compared with the N-terminaltails of GPCRs endowed with a signal peptide. This suggeststhat extracellular N-terminal tails of eukaryotic plasma membraneproteins may be translocated by different mechanisms dependingon whether or not they are preceded by a signal peptide.