Allosteric formulation of thermal transitions in macromolecules, including effects of ligand binding and oligomerization

We examine the effects of concentration (aggregation), buffers, and ligation, under conditions of either constant ligand activity or limited total amount of ligand, upon thermal denaturation of macromolecules as measured by scanning calorimetry. In doing so we utilize and extend an earlier generalized allosteric treatment [S. J. Gill, B. Richey, G. Bishop, and J. Wyman (1985) Biophys. Chem. 21, 1-14], applicable to ligand binding, enthalpy changes, and volume changes in a macromolecular system. The approach is contrasted with formulations based on the idea of structural domains. We show how information from the full scanning calorimetric curves can be utilized in arriving at and testing appropriate models for observed behavior in selected examples.     

HDX‐MS reveals orthosteric and allosteric changes in apolipoprotein‐D structural dynamics upon binding of progesterone

Apolipoprotein‐D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein‐D adopts an eight‐stranded β‐barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α‐helix. Crystallography studies of recombinant apolipoprotein‐D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX‐MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X‐ray crystallography, making HDX‐MS an invaluable orthogonal technique. Here, we report an HDX‐MS protocol for apolipoprotein‐D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand‐free apolipoprotein‐D revealed apolipoprotein‐D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein‐D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N‐terminus and C‐terminus. Together, our experiments provide insight into apolipoprotein‐D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein‐D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein‐D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein‐D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein‐D structure from X‐ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein‐D, to elucidate how proteins change flexibility when binding to small molecules.     

Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange

The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIalpha) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIalpha) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an alpha-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIalpha and RIIalpha D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIalpha compared to RIIalpha, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIalpha indicated that there were more binding constraints for the AKB ligand when bound to RIalpha. This was in contrast to RIIalpha, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIalpha AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.     

Promoting RNA helical stacking via A-minor junctions

RNA molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3D architectures. The A-minor junction is a class of RNA motifs that specifically controls coaxial stacking of helices in natural RNAs. A sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified A-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. This class of modular motifs follows a topological rule that can accommodate a variety of interchangeable A-minor interactions with distinct local structural motifs. Overall, two different types of A-minor junctions can be distinguished based on their functional self-assembling behavior: one group makes use of triloops or GNRA and GNRA-like loops assembling with helices, while the other takes advantage of more complex tertiary receptors specific for the loop to gain higher stability. This study demonstrates how different structural motifs of RNA can contribute to the formation of topologically equivalent helical stacks. It also exemplifies the need of classifying RNA motifs based on their tertiary structural features rather than secondary structural features. The A-minor junction rule can be used to facilitate tertiary structure prediction of RNAs and rational design of RNA parts for nanobiotechnology and synthetic biology.     

Molecular dynamics simulations of the ligand-binding domain of the ionotropic glutamate receptor GluR2.

Ionotropic glutamate receptors are essential for fast synaptic nerve transmission. Recent x-ray structures for the ligand-binding (S1S2) region of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive receptor have suggested how differences in protein/ligand interactions may determine whether a ligand will behave as a full agonist. We have used multiple molecular dynamics simulations of 2-5 ns duration to explore the structural dynamics of GluR2 S1S2 in the presence and absence of glutamate and in a complex with kainate. Our studies indicate that not only is the degree of domain closure dependent upon interactions with the ligand, but also that protein/ligand interactions influence the motion of the S2 domain with respect to S1. Differences in domain mobility between the three states (apo-S1S2, glutamate-bound, and kainate-bound) are surprisingly clear-cut. We discuss how these changes in dynamics may provide an explanation relating the mechanism of transmission of the agonist-binding event to channel opening. We also show here how the glutamate may adopt an alternative mode of binding not seen in the x-ray structure, which involves a key threonine (T480) side chain flipping into a new conformation. This new conformation results in an altered pattern of hydrogen bonding at the agonist-binding site.     

Small-angle X-ray scattering studies of enzymes

Enzyme function requires conformational changes to achieve substrate binding, domain rearrangements, and interactions with partner proteins, but these movements are difficult to observe. Small-angle X-ray scattering (SAXS) is a versatile structural technique that can probe such conformational changes under solution conditions that are physiologically relevant. Although it is generally considered a low-resolution structural technique, when used to study conformational changes as a function of time, ligand binding, or protein interactions, SAXS can provide rich insight into enzyme behavior, including subtle domain movements. In this perspective, we highlight recent uses of SAXS to probe structural enzyme changes upon ligand and partner-protein binding and discuss tools for signal deconvolution of complex protein solutions.     

Stereospecific effects of ascorbic acid and analogues on D1 and D2 agonist binding.

Ascorbic acid inhibited the specific binding of both the D1 agonist, [3H] SKF 38393, and the D2 agonist, [3H] N-0437 at physiologically relevant concentrations. This inhibition was both stereospecific and receptor selective. Using ligand concentrations approximating their KD's, the IC50's for ascorbate and two structural analogues, isoascorbate and D-glucoascorbate, were determined. The rank order of IC50's at both D1 and D2 were D-glucoascorbate greater than isoascorbate greater than ascorbate. However, the IC50 for each compound was greater at D1 than D2. Evaluation of the relationship between the IC50 for ascorbate and the ligand concentration using both the D1 and the D2 ligand yielded data inconsistent with competitive inhibition models. Preliminary experiments were conducted to evaluate the site and type of inhibition with results consistent with an allostearic effect at the level of the receptor.     

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism. Instead, we characterize the cleft-closing transition as a load and lock mechanism. We find that when two cross-cleft hydrogen bonds are disrupted the protein undergoes a rapid cleft opening transition. At the same time, the dynamical behavior of the cleft in the presence of the glutamate ligand is only weakly affected by the S652 peptide bond in its flipped conformation observed in the crystal structure. The residue E705 plays significant role in stabilization of the closed conformation via electrostatic interactions. The presence of the E705-K730 salt bridge seems to correlate strongly withthe cleft opening transition in the MD simulations.     

Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin

Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D(140)XD(142)XE(144) sequence motif. In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced k(cat) and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with k(cat), indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue.     

What is the relationship between the global structures of apo and holo proteins?

It is well known that ligand binding and release may induce a wide range of structural changes in a receptor protein, varying from small movements of loops or side chains in the binding pocket to large‐scale domain hinge‐bending and shear motions or even partial unfolding that facilitates the capture and release of a ligand. An interesting question is what in general are the conformational changes triggered by ligand binding? The aim of this work is analyze the magnitude of structural changes in a protein resulting from ligand binding to assess if the state of ligand binding needs to be included in template‐based protein structure prediction algorithms. To address this issue, a nonredundant dataset of 521 paired protein structures in the ligand‐free and ligand‐bound form was created and used to estimate the degree of both local and global structure similarity between the apo and holo forms. In most cases, the proteins undergo relatively small conformational rearrangements of their tertiary structure upon ligand binding/release (most root‐mean‐square‐deviations from native, RMSD, are <1 Å). However, a clear difference was observed between single‐ and multiple‐domain proteins. For the latter, RMSD changes greater than 1 Å and sometimes larger were found for almost 1/3 of the cases; these are mainly associated with large‐scale hinge‐bending movements of entire domains. The changes in the mutual orientation of individual domains in multiple‐domain proteins upon ligand binding were investigated using a mechanistic model based on mass‐weighted principal axes as well as interface buried surface calculations. Some preferences toward the anticipated mechanism of protein domain movements are predictable based on the examination of just the ligand‐free structural form. These results have applications to protein structure prediction, particularly in the context of protein domain assembly, if additional information concerning ligand binding is exploited. Proteins 2008. © 2007 Wiley‐Liss, Inc.     

Light-induced FTIR difference spectroscopy of the S(2)-to-S(3) state transition of the oxygen-evolving complex in Photosystem II

We have applied flash-induced FTIR spectroscopy to study structural changes upon the S(2)-to-S(3) state transition of the oxygen-evolving complex (OEC) in Photosystem II (PSII). We found that several modes in the difference IR spectrum are associated with bond rearrangements induced by the second laser flash. Most of these IR modes are absent in spectra of S(2)/S(1), of the acceptor-side non-heme ion, of Yradical(D)/Y(D) and of S(3)'/S(2)' from Ca-depleted PSII preparations. Our results suggest that these IR modes most likely originate from structural changes in the oxygen-evolving complex itself upon the S(2)-to-S(3) state transition in PSII.     

Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, K_D, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (K_D ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD).     

Human flap endonuclease-1: conformational change upon binding to the flap DNA substrate and location of the Mg2+ binding site

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is a key enzyme in DNA replication and repair. FEN-1 recognizes the 5'-flap DNA structure and cleaves it, a specialized endonuclease function essential for the processing of Okazaki fragments during DNA replication and for the repair of 5'-end single-stranded tails from nicked double-stranded DNA substrates. Magnesium is a cofactor required for nuclease activity. We have used Fourier transform infrared (FTIR) spectroscopy to better understand how Mg2+ and flap DNA interact with human FEN-1. FTIR spectroscopy provides three fundamentally new insights into the structural changes induced by the interaction of FEN-1 with substrate DNA and Mg2+. First, FTIR difference spectra in the amide I vibrational band (1600-1700 cm(-1)) reveal a change in the secondary structure of FEN-1 induced by substrate DNA binding. Quantitative analysis of the FTIR spectra indicates a 4% increase in helicity upon DNA binding or about 14 residues converted from disordered to helical conformations. The observation that the residues are disordered without DNA strongly implicates the flexible loop region. The conversion to helix also suggests a mechanism for locking the flexible loop region around the bound DNA. This is the first direct experimental evidence for a binding mechanism that involves a secondary structural change of the protein. Second, in contrast with DNA binding, no change is observed in the secondary structure of FEN-1 upon Mg2+ binding to the wild type or to the noncleaving D181A mutant. Third, the FTIR results provide direct evidence (via the carboxylate ligand band at 1535 cm(-1)) that not only is D181 a ligand to Mg2+ in the human enzyme but Mg2+ binding does not occur in the D181A mutant which lacks this ligand.     

Agonist-independent and -dependent oligomerization of dopamine D(2) receptors by fusion to fluorescent proteins.

Oligomerization of the short (D(2S)) and long (D(2L)) isoforms of the dopamine D(2) receptor was explored in transfected Cos-7 cells by their C-terminal fusion to either an enhanced cyan or enhanced yellow fluorescent protein (ECFP or EYFP) and the fluorescent fusion protein interaction was monitored by a fluorescence resonance energy transfer (FRET) assay. The pharmacological properties of the fluorescent fusion proteins, as measured by both displacement of [(3)H]nemonapride binding and agonist-mediated stimulation of [(35)S]GTPgammaS binding upon co-expression with a G(alphao)Cys(351)Ile protein, were not different from the respective wild-type D(2S) and D(2L) receptors. Co-expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:EYFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% in the EYFP-specific fluorescent signal. These data are consistent with a close proximity of both D(2S) and D(2L) receptor pairs of fluorescent fusion proteins in the absence of ligand. The agonist-independent D(2S) receptor oligomerization could be attenuated by co-expression with either a wild-type, non-fluorescent D(2S) or D(2L) receptor subtype, but not with a distinct beta(2)-adrenoceptor. Incubation with the agonist (-)-norpropylapomorphine dose-dependently (EC(50): 0.23+/-0.06 nM) increased the FRET signal for the co-expression of D2S:ECFP and D2S:EYFP, in support of agonist-dependent D(2S) receptor oligomerization. In conclusion, our data strongly suggest the occurrence of dopamine D(2) receptor oligomers in intact Cos-7 cells.     

High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy

We describe here the high-level expression of bovine trypsinogen in E. coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium. Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy. 2D [(1)H-(15)N]-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation. Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa). Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis. These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation. In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure. This was true both for S195A and for S195A/D189S trypsins. This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.     

Structural changes and enhancements in DNase I footprinting experiments.

In footprinting experiments, an increase in DNA cleavage with addition of ligand to a system may be due to a ligand-induced structural change. Ligand binding also enhances cleavage by displacing the cleavage agent from ligand-binding sites, thus increasing its concentration elsewhere. The theory and characteristics of this mass-action enhancement are given, and it is shown how it may be recognized. Results of DNase I footprinting of small oligomers, with actinomycin D as ligand, are analyzed to reveal which enhancements are due to mass action, and which can reasonably be ascribed to structural changes. Patterns in the footprinting plots from our experiments on actinomycin D binding to a 139-base-pair DNA fragment (with DNase I as a probe) are studied in the same way. The likely origins of these patterns are discussed, as are enhancements occurring with other probes commonly used in footprinting experiments.     

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.     

A comparative analysis of the triloops in all high-resolution RNA structures reveals sequence structure relationships

Despite an increasing number of experimentally determined RNA structures, the gap between the number of structures and that of RNA families is still growing. To overcome this limitation, efficient and reliable RNA modeling methodologies must be developed. In order to reach this goal, here, we show how triloop sequence-structure relationships have been inferred through a systematic analysis of all triloops found in available high-resolution structures. The structural annotation of all triloops allowed us to define discrete states of the triloop's conformational space, and therefore an explicit sequence-to-structure relation. The sequence-structure relationships inferred from this explicit relation are presented in a convenient modeling table that provides a limited set of possible three-dimensional structures given any triloop sequence. The table is indexed by the two nucleotides that form the triloop's flanking base pair, since they are shown to provide the most information about the triloop three-dimensional structures. We also report the observations in the X-ray crystallographic structures of important conformational variations, which we believe might be the result of RNA dynamic.     

Volume of Hsp90 ligand binding and the unfolding phase diagram as a function of pressure and temperature

Volume changes that accompany protein unfolding and ligand binding are important but largely neglected thermodynamic parameters that may facilitate rational drug design. Here, we determined the volume of lead compound ICPD47 binding to an anticancer target, heat shock protein 90 N-terminal domain, using a pressure shift assay (PressureFluor). The ligand exhibited a stabilizing effect on the protein by increasing its melting pressure and temperature. The Gibbs free energy of unfolding depends on the absence or presence of ligand and has an elliptical shape. Ellipse size increases upon addition of the strongly binding ligand, which stabilizes the protein. The three-dimensional (3D) ellipsoidal surface of the Gibbs free energy of unfolding was calculated with increasing ligand concentrations. The negative volume of ligand binding was relatively large and significantly exceeded the volume of protein unfolding. The pressure shift assay technique could be used to determine the volume changes associated with both protein unfolding as well as ligand binding to protein.