Classification and annotation of the relationship between protein structural change and ligand binding

The causal relationship between protein structural change and ligand binding was classified and annotated for 839 nonredundant pairs of crystal structures in the Protein Data Bank—one with and the other without a bound low-molecular-weight ligand molecule. Protein structural changes were first classified into either domain or local motions depending on the size of the moving protein segments. Whether the protein motion was coupled with ligand binding was then evaluated based on the location of the ligand binding site and by application of the linear response theory of protein structural change. Protein motions coupled with ligand binding were further classified into either closure or opening motions. This classification revealed the following: (i) domain motions coupled with ligand binding are dominated by closure motions, which can be described by the linear response theory; (ii) local motions frequently accompany order-disorder or α-helix-coil conformational transitions; and (iii) transferase activity (Enzyme Commission   number 2) is the predominant function among coupled domain closure motions. This could be explained by the closure motion acting to insulate the reaction site of these enzymes from environmental water.     

Tight binding ligand approach to oligosaccharide-grafted protein

A novel type of artificial glycoprotein was developed, by using dihydrofolate reductase (DHFR) and methotrexate (MTX) as a protein-ligand pair. Various oligosaccharides linked to MTX were shown to bind tightly with DHFR and afforded oligosaccharide-grafted protein, which could be isolated easily by lectin beads.     

Free energy simulations for protein ligand binding and stability

Abstract

We summarize several computational techniques to determine relative free energies for condensed-phase systems. The focus is on practical considerations which are capable of making direct contact with experiments. Particular applications include the thermodynamic stability of apo- and holo-myoglobin, insulin dimerization free energy, ligand binding in lysozyme, and ligand diffusion in globular proteins. In addition to provide differential free energies between neighboring states, converged umbrella sampling simulations provide insight into migration barriers and ligand dissociation barriers and analysis of the trajectories yield additional insight into the structural dynamics of fundamental processes. Also, such simulations are useful tools to quantify relative stability changes for situations where experiments are difficult. This is illustrated for NO-bound myoglobin. For the dissociation of benzonitrile from lysozyme it is found that long umbrella sampling simulations are required to approximately converge the free energy profile. Then, however, the resulting differential free energy between the bound and unbound state is in good agreement with estimates from molecular mechanics with generalized Born surface area simulations. Furthermore, comparing the barrier height for ligand escape suggests that ligand dissociation contains a non-equilibrium component.     

Computational study of ligand binding to protein receptors

We have determined, for the first time, the enthalpic contributions to the energy change associated with ligand reorganization (LR) upon the binding of the same ligand to multiple sites within human serum albumine (HSA). Quantum mechanics based density functional theory (DFT) has been used for the LR calculations, which provides much better accuracy than previously used molecular mechanics methods (MM). Our findings show that for some ligands these enthalpic contributions can be attributed to specific structural and conformational changes.     

Relationships between ligand binding sites, protein architecture and correlated paths of energy and conformational fluctuations

The statistical thermodynamics basis of energy and residue position fluctuations is explained for native proteins. The protein and its surroundings are treated as a canonical system with emphasis on the effects of energy exchange between the two. Fluctuations of the energy are related to fluctuations of residue positions, which in turn are related to the connectivity matrix of the protein, thus establishing a connection between energy fluctuation pathways and protein architecture. The model gives the locations of hotspots for ligand binding and identifies the pathways of energy conduction within the protein. Results are discussed in terms of two sets of models, the BPTI and 12 proteins that contain the PDZ domain. A possible use of the model for determining functionally similar domains in a diverse set of proteins is pointed out.     

Stabilization of the FK506 binding protein by ligand binding.

Although the rotamase activity of the FK506 binding protein is inhibited by ligand binding, it is hypothesized that the ligand/protein complex itself may be responsible for the immunosuppressive effects of FK506. We have therefore examined the structure of the FK506 binding protein in the presence of an analog of FK506 (FK520) by a combination of fluorescence, CD, FTIR and calorimetry. While only small changes in the overall structure of the protein may be induced by ligand, a large change in thermal stability of the binding protein is observed.     

Multivalent ligand binding by serum mannose-binding protein.

The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP.     

Determinants of ligand binding to cAMP-dependent protein kinase

Protein kinases are essential for the regulation of cellular growth and metabolism. Since their dysfunction leads to debilitating diseases, they represent key targets for pharmaceutical research. The rational design of kinase inhibitors requires an understanding of the determinants of ligand binding to these proteins. In the present study, a theoretical model based on continuum electrostatics and a surface-area-dependent nonpolar term is used to calculate binding affinities of balanol derivatives, H-series inhibitors, and ATP analogues toward the catalytic subunit of cAMP-dependent protein kinase (cAPK or protein kinase A). The calculations reproduce most of the experimental trends and provide insight into the driving forces responsible for binding. Nonpolar interactions are found to govern protein-ligand affinity. Hydrogen bonds represent a negligible contribution, because hydrogen bond formation in the complex requires the desolvation of the interacting partners. However, the binding affinity is decreased if hydrogen-bonding groups of the ligand remain unsatisfied in the complex. The disposition of hydrogen-bonding groups in the ligand is therefore crucial for binding specificity. These observations should be valuable guides in the design of potent and specific kinase inhibitors.     

The ligand binding profiles of estrogen receptors alpha and beta are species dependent

Estrogens and selective estrogen receptor modulators are used for the treatment and prevention of conditions resulting from menopause. Since estrogens exert their activity by binding to nuclear receptors, there is intense interest in developing new ligands for the two known estrogen receptor subtypes, ER-alpha and ER-beta. Characterization assays used to profile new estrogen receptor ligands often utilize receptors from different species, with the assumption that they behave identically. To test this belief, we have profiled a number of estrogens, other steroids, phytoestrogens and selective estrogen receptor modulators in a solid phase radioligand binding assay using recombinant protein for human, rat, and mouse ER-alpha and ER-beta. Certain compounds show species dependent binding preferences for ER-alpha or ER-beta, leading to differences in receptor subtype selectivity. The amino acids identified by crystallography as lining the ligand binding cavity are the same among the three species, suggesting that as yet unidentified amino acids contribute to the structure of the binding site. We conclude from this analysis that the ability of a compound to selectively bind to a particular ER subtype can be species dependent.     

The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH 7.4 (pI = 7–8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 °C) and molar enthalpy (146 kcal M^− 1) values increasing to 78 °C and 163 kcal M^− 1 at 10 μM holo-FBP, while those of apo-FBP were 55 °C and 105 kcal M^− 1. Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10 μM) in DLS after a step-wise rise in temperature to 78 °C ≈ Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH 5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH 5 (Tm 38 °C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15 M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.     

Studies on ligand binding to histidine triad nucleotide binding protein 1

Histidine triad nucleotide binding protein (HINT1) is an intracellular protein that binds purine mononucleotides. Strong sequence conservation suggests that these proteins play a fundamental role in cell biology, however its exact cellular function continues to remain elusive. nuclear magnetic resonance (NMR) studies using STD and HSQC were conducted to observe ligand binding to HINT1. These studies were confirmed using fluorescence spectroscopy titrations. We found that AICAR, the first non-phosphate containing ligand, binds to mouse histidine triad nucleotide binding protein 1 (HINT1). Chemical shift perturbations are mapped onto the X-ray structure showing AICAR binds at the same site as GMP. The NMR results demonstrated that this method will be valuable for the future screening of small molecules that can be used to modulate the function of HINT1.     

Mapping the ligand binding pocket in the cellular retinaldehyde binding protein

Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Altered UV-visible spectra and lower retinoid binding affinities were observed for the mutants, supporting modified ligand interactions. Altered kinetic parameters were observed for RDH5 oxidation of 11-cis-retinol bound to rCRALBP mutants M222A, M225A, and W244F, supporting impaired substrate carrier function. Heteronuclear single quantum correlation NMR analyses confirmed localized structural changes upon photoisomerization of rCRALBP-bound 11-cis-retinal and demonstrated ligand-dependent conformational changes for residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore, residues Met-208, Met-222, Met-225, and Trp-244 are within a region exhibiting high homology to the ligand binding cavity of phosphatidylinositol transfer protein. Overall the data implicate Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the CRALBP ligand binding cavity.     

Microcalorimetric indications for ligand binding as a function of the protein for galactoside-specific plant and avian lectins

The process cascade leading to the final accommodation of the carbohydrate ligand in the lectin's binding site comprises enthalpic and entropic contributions of the binding partners and solvent molecules. With emphasis on lactose, N-acetyllactosamine, and thiodigalactoside as potent inhibitors of binding of galactoside-specific lectins, the question was addressed to what extent these parameters are affected as a function of the protein. The microcalorimetric study of carbohydrate association to the galectin from chicken liver (CG-16) and the agglutinin from Viscum album (VAA) revealed enthalpy-entropy compensation with evident protein type-dependent changes for N-acetyllactosamine. Reduction of the entropic penalty by differential flexibility of loops or side chains and/or solvation properties of the protein will have to be reckoned with to assign a molecular cause to protein type-dependent changes in thermodynamic parameters for lectins sharing the same monosaccharide specificity.     

Model assembly study of the ligand binding by p-hydroxybenzoate hydroxylase: Correlation between the calculated binding energies and the experimental dissociation constants

The energies of binding of seven ligands by p-hydroxybenzoate hydroxylase (PHBH) were calculated theoretically. Direct enzyme–ligand interaction energies were calculated using the ab initio quantum mechanical model assembly of the active site at the 3-21G level. Solvation energies of the ligands needed in the evaluation of the binding energies were calculated with the semiempirical AM1–SM2 method and the long-range electrostatic interaction energies between the ligands and the protein matrix classically using the static charge distributions of the ligands and the protein. Energies for proton-transfer between the ligands OH or SH substituent at position 4 and the active-site tyrosine within the ab initio model assemblies were calculated and compared to the corresponding pK_as in aqueous solution. Excluding 3,4-dihydroxybenzoate, the natural product of PHBH, a linear relationship between the calculated binding energies and the experimental binding free energies was found with a correlation coefficient of 0.90. Contributions of the direct enzyme–ligand interaction energies, solvation energies and the long-range electrostatic interaction energies to the calculated binding energies were analyzed. The proton-transfer energies of the ligands with substituents ortho to the ionized OH were found to be perturbed less in the model calculations than the energies of their meta isomers as deduced from the corresponding pK_as. © 1995 Wiley-Liss, Inc.     

Sampling and energy evaluation challenges in ligand binding protein design

The steroid hormone 17α‐hydroxylprogesterone (17‐OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17‐OHP containing an extended, nonpolar, shape‐complementary binding pocket for the four‐ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17‐OHP with micromolar affinity. A co‐crystal structure of one of the designs revealed that 17‐OHP is rotated 180° around a pseudo‐two‐fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same “flipped” orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two‐fold symmetry of the molecule.     

Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs.