Effect of variation of the strength of the aromatic interactions of tryptophan on the cooperative structural refolding behavior of a peptide from HIV 1

A 15-residue sequence (LPCRIKQFINMWQEV) forming the principal CD4-binding domain of gp120 from HIV 1 displays unusual, highly cooperative refolding from β-hairpin to 3₁₀ helix when the polarity of the surrounding medium drops below a critical point, the so-called conformational switch. The tryptophan at position 12 has been shown to be essential for the cooperativity of the refolding process, and several lines of evidence from earlier work had suggested that it was the aromatic quadrupole that was responsible for this. To define more precisely what physico-chemical properties of tryptophan brought about the unique behavior of this peptide, nonproteogenic aromatic amino acids have been selected based on desired alterations in quadrupole moment, electrostatic potential surface, and binding energy to ions. These were built into the peptide in the place of tryptophan and their effect on switch behavior examined. It could be shown that a minimal strength of the quadrupole moment is necessary but not sufficient to enforce cooperativity of refolding, with other properties of tryptophan playing a role in the optimum interaction of this residue with other side chains of the peptide.     

Galpha Gbetagamma dissociation may be due to retraction of a buried lysine and disruption of an aromatic cluster by a GTP-sensing Arg Trp pair

The heterotrimeric G protein alpha subunit (Galpha) functions as a molecular switch by cycling between inactive GDP-bound and active GTP-bound states. When bound to GDP, Galpha interacts with high affinity to a complex of the beta and gamma subunits (Gbetagamma), but when bound to GTP, Galpha dissociates from this complex to activate downstream signaling pathways. Galpha's state is communicated to other cellular components via conformational changes within its switch I and II regions. To identify key determinants of Galpha's function as a signaling pathway molecular switch, a Bayesian approach was used to infer the selective constraints that most distinguish Galpha and closely related Arf family GTPases from distantly related translational and metabolic GTPases. The strongest of these constraints are imposed on seven residues within or near the switch II region. Likewise, constraints imposed on Galpha but not on other, closely related molecular switches correspond to four nearby residues. These constraints are explained by a proposed mechanism for GTP-induced dissociation of Galpha from Gbetagamma where an Arg-Trp pair senses the presence of bound GTP leading to conformational retraction of a nearby lysine and to disruption of an aromatic cluster. Within a complex of Gialpha, Gibetagamma, and GDP, this lysine establishes greater surface contact with Gibeta than does any other residue in Gialpha, whereas the aromatic cluster packs against a highly conserved tryptophan in Gibeta that establishes greater surface contact with Gialpha than does any other residue in Gibeta. Other structural features associated with Galpha functional divergence further support the proposed mechanism.     

Molecular simulations of mutually exclusive folding in a two-domain protein switch

A major challenge with testing designs of protein conformational switches is the need for experimental probes that can independently monitor their individual protein domains. One way to circumvent this issue is to use a molecular simulation approach in which each domain can be directly observed. Here we report what we believe to be the first molecular simulations of mutually exclusive folding in an engineered two-domain protein switch, providing a direct view of how folding of one protein drives unfolding of the other in a barnase-ubiquitin fusion protein. These simulations successfully capture the experimental effects of interdomain linker length and ligand binding on the extent of unfolding in the less stable domain. In addition, the effect of linker length on the potential for oligomerization, which eliminates switch activity, is in qualitative agreement with analytical ultracentrifugation experiments. We also perform what we believe to be the first study of protein unfolding via progressive localized compression. Finally, we are able to explore the kinetics of mutually exclusive folding by determining the effect of linker length on rates of unfolding and refolding of each protein domain. Our results demonstrate that molecular simulations can provide seemingly novel biological insights on the behavior of individual protein domains, thereby aiding in the rational design of bifunctional switches.     

Classification of doubly wound nucleotide binding topologies using automated loop searches

A classification is presented of doubly wound α/β nucleotide binding topologies, whose binding sites are located in the cleft formed by a topological switch point. In particular, the switch point loop nearest the N-terminus is used to identify specific structural classes of binding protein. This yields seven structurally distinct loop conformations, which are subsequently used as motifs for scanning the Protein Data Bank. The searches, which are effective at identifying functional relationships within a large database of structures, reveal a remarkable and previously unnoticed similarity between the coenzyme binding sites of flavodoxin and tryptophan synthetase, even though there is no sequence or topological similarity between them.     

Structural basis for a change in substrate specificity: crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K(M)) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg(2+) to 2.0 A resolution. On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The S113E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site. In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the S113E substitution and the binding of substrates act together to promote catalysis of isopropylmalate. Ligand binding results in reorientation of the active site helix that contains residues 113 through 116. E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP. In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specificity.     

Solvent polarity-dependent structural refolding: A CD and NMR study of a 15 residue peptide

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a β-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular β-sheet to a helical secondary structure is known as “switch” behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the “switch.” The tetrad has a strong β-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3₁₀-helix suggesting that the tetrad adopts a type III β-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3₁₀-helix at a critical polarity favoring intrachain hydrogen bonds. © 1995 Wiley-Liss, Inc.     

Role of the C-terminal helix 9 in the stability and ligandin function of class alpha glutathione transferase A1-1

Helix 9 at the C-terminus of class alpha glutathione transferase (GST) polypeptides is a unique structural feature in the GST superfamily. It plays an important structural role in the catalytic cycle. Its contribution toward protein stability/folding as well as the binding of nonsubstrate ligands was investigated by protein engineering, conformational stability, enzyme activity, and ligand-binding methods. The helix9 sequence displays an unfavorable propensity toward helix formation, but tertiary interactions between the amphipathic helix and the GST seem to contribute sufficient stability to populate the helix on the surface of the protein. The helix's stability is enhanced further by the binding of ligands at the active site. The order of ligand-induced stabilization increases from H-site occupation, to G-site occupation, to the simultaneous occupation of H- and G-sites. Ligand-induced stabilization of helix9 reduces solvent accessible hydrophobic surface by facilitating firmer packing at the hydrophobic interface between helix and GST. This stabilized form exhibits enhanced affinity for the binding of nonsubstrate ligands to ligandin sites (i.e., noncatalytic binding sites). Although helix9 contributes very little toward the global stability of hGSTA1-1, its conformational dynamics have significant implications for the protein's equilibrium unfolding/refolding pathway and unfolding kinetics. Considering the high concentration of reduced glutathione in human cells (about 10 mM), the physiological form of hGSTA1-1 is most likely the thiol-complexed protein with a stabilized helix9. The C-terminus region (including helix9) of the class alpha polypeptide appears not to have been optimized for stability but rather for catalytic and ligandin function.     

Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.     

Structural transitions in the protein L denatured state ensemble

We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.     

A dynamic N-capping motif in cytochrome b5: evidence for a pH-controlled conformational switch

Apocytochrome b5 is a marginally stable protein exhibiting under native conditions a slow conformational exchange in its C-terminal region. The affected elements of secondary structure include a 3(10)-helix containing at its N-terminus a histidine Ncap and a subsequent proline. Participation of the neutral histidine side-chain in backbone amide capping lowers the imidazole pKa. To explore the nature of the conformational exchange in the protein and determine whether it is related to cis-trans isomerization of the His-Pro bond, three octapeptides encompassing the helix were synthesized and studied by NMR spectroscopy. One corresponded to the wild-type sequence, the second was the D-histidine epimer, and the third contained an alanine in place of the proline. It was found that the rates of cis-trans interconversion in the proline-containing peptides were slower than the rates of the conformational exchange in the protein. In addition, the wild-type peptide hinted at a predisposition for Ncap formation when in the trans configuration. Analysis of the pH response of the peptides and protein suggested that at pH near neutral, the conformational exchange detected in the protein involved only species with a trans His-Pro bond and could be approximated with a three-state model by which the terminal helix sampled a locally unfolded state. This state, which contained an uncapped histidine with a normal pKa, partitioned into neutral and protonated populations according to pH. The intrinsic conformational bias of the wild-type peptide and the pH-driven equilibria illustrated how a 3(10)-element could serve as a nucleation site for structural rearrangement.     

Conformational transitions of thioredoxin in guanidine hydrochloride

Spectral and hydrodynamic measurements of thioredoxin from Escherichia coli indicate that the compact globular structure of the native protein is significantly unfolded in the presence of guanidine hydrochloride concentrations in excess of 3.3 M at neutral pH and 25 degrees C. This conformational transition having a midpoint at 2.5 M denaturant is quantitatively reversible and highly cooperative. Stopped-flow measurements of unfolding in 4 M denaturant, observed with tryptophan fluorescence as the spectral probe, reveal a single kinetic phase having a relaxation time of 7.1 +/- 0.2 s. Refolding measurements in 2 M denaturant reveal three kinetic phases having relaxation times of 0.54 +/- 0.23, 14 +/- 6, and 500 +/- 130 s, accounting for 12 +/- 2%, 10 +/- 1%, and 78 +/- 3% of the observed change in tryptophan fluorescence. The dominant slowest phase is generated in the denatured state with a relaxation time of 42 s observed in 4 M denaturant. Both the slowest phase observed in refolding and the generation of the slowest phase in the denatured state have an activation enthalpy of 22 +/- 1 kcal/mol. These features of the slowest phase are compatible with an obligatory peptide isomerization of proline-76 to its cis isomer prior to refolding.     

LRRK2 dynamics analysis identifies allosteric control of the crosstalk between its catalytic domains

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease–related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2_RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a “cap” that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

The Parkinson’s disease-related protein LRRK2 contains the two major molecular switches in biology; a kinase and a GTPase. This study uses hydrogen-deuterium exchange mass-spectrometry and molecular dynamics simulations to explore the conformational space of the four C-terminal domains of LRRK2, highlighting two essential regulatory helices that control LRRK2 dynamics.     

Equilibrium and kinetic measurements of the conformational transition of thioredoxin in urea

Addition of urea to solutions of Escherichia coli thioredoxin results in a cooperative unfolding of the protein centered at 6.7 M urea at 25 degrees C and 5.1 M urea at 2 degrees C and neutral pH as judged by changes in tryptophan fluorescence emission, far-ultraviolet circular dichroism, and exclusion chromatography. Kinetic profiles of changes in tryptophan fluorescence emission intensity were analyzed following either manual or stopped-flow mixing to initiate unfolding or refolding. Unfolding of the native protein occurs in a single kinetic phase whose time constant is markedly dependent on urea concentration. Refolding of the urea-denatured protein occurs in a multiplicity of kinetic phases whose time constants and fractional amplitudes are also dependent upon urea concentration. Urea gradient gel electrophoretic and exclusion chromatographic measurements suggest the transient accumulation of at least one and likely two compact nativelike intermediate conformations during refolding. Simulations of both electrophoretic and chromatographic results suggest that the intermediate conformations are generated by the concerted action of the middle and fast refolding phases.     

Relocation or duplication of the helix A sequence of T4 lysozyme causes only modest changes in structure but can increase or decrease the rate of folding

In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associated with the C-terminal domain in the folded structure. To investigate the implications of this arrangement for the folding of the protein, we first created a circularly permuted variant with a new amino terminus at residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminus of the molecule. The protein crystallized nonisomorphously with the wild type but has a very similar structure, showing that the unit consisting of helix A and the C-terminal domain can be reconstituted from a contiguous polypeptide chain. The protein is less stable than the wild type but folds slightly faster. We then produced a second variant in which the helix A sequence was appended at the C-terminus (as in the first variant), but was also restored at the N-terminus (as in the wild type). This variant has two helix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete for the same site in the folded protein. The crystal structure shows that it is the N-terminal sequence that folds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loop out. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantly slower. The reduction in rate is attributed to the presence of the two identical sequence segments which compete for a single, mutually exclusive, site.     

Insertion of a chaperone domain converts FKBP12 into a powerful catalyst of protein folding

The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra "insert-in-flap" or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module. A prolyl isomerase catalytic site and a separate chaperone site with an adapted affinity for refolding protein chains are the key elements for a productive coupling between the catalysis of prolyl isomerization and conformational folding in the enzymatic mechanisms of SlyD and other prolyl isomerases, such as trigger factor and FkpA.     

The Charge-dipole Pocket: A Defining Feature of Signaling Pathway GTPase On/Off Switches

Ras-like GTPases function as on/off switches in intracellular signaling pathways. Their on or off state is communicated through conformational changes in the so-called switch I and II regions. It is commonly believed that the distinguishing molecular features of these GTPases are well known. Here, however, I identify—through a Bayesian iterative analysis of GTPase evolutionary divergence—a previously undescribed switch II structural component that (along with previously described, functionally critical residues) most distinguish these signaling pathway on/off switches from other GTPases. In certain Ras-like GTPases this newly-identified component forms an aromatic pocket around the negative-dipole moment at the end of a switch II helix with a positively charged residue inserted into the pocket. This helix is oriented in a specific direction away from the GTPase core, but is reoriented dramatically upon disruption of the charge-dipole pocket. The charge-dipole pocket occurs in both the on and off states and both the charge-dipole pocket and an alternative configuration occur within the unit cell of a single crystal structure of Rab5a GTPase in the off state. Thus, the charge-dipole pocket configuration is closely associated, not with the on or off state, but rather with formation of the outward-oriented helix and, as a result, with restructuring of the switch II N-terminal region, which has a critical role both in sensing the on/off state and in mediating GTP hydrolysis and nucleotide exchange.     

Measurements of label free protein concentration and conformational changes using a microfluidic UV-LED method

This paper presents a microchip-based system for measuring concentrations and dynamic conformational changes in proteins without any use of extrinsic fluorescent labeling. The microchannel flow of protein molecules was integrated with an ultraviolet light-emitting diode (UV-LED, lambda ex = 295 nm) and a photodetector (lambda em = 330 nm). The intrinsic fluorescence shift, arising from selectively exciting aromatic amino acid tryptophan (Trp), was monitored to quantify refolding pathways by dynamically varying the concentration of the chemical denaturant, urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls, rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. The concentrations of proteins were first measured using the UV-LED microfluidic platform, and the data showed detection limits down to 72, 128, and 250 nM for tryptophan, bovine serum albumin (BSA), and bovine carbonic anhydrase (BCA), respectively. To validate the protein assay method, folding transition experiments were performed using a well-characterized protein, BSA. The microchip protein refolding transitions using intrinsic fluorescence were well-correlated with conventional fluorometer experiments. The microfluidic platform facilitates refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material. The technique offers a real alternative to bulky microfluidic systems consisting of large and expensive laser-based designs.