Chemical basis of glycine riboswitch cooperativity

The glycine binding riboswitch forms a unique tandem aptamer structure that binds glycine cooperatively. We employed nucleotide analog interference mapping (NAIM) and mutagenesis to explore the chemical basis of glycine riboswitch cooperativity. Based on the interference pattern, at least two sites appear to facilitate cooperative tertiary interactions, namely, the minor groove of the P1 helix from aptamer 1 and the major groove of the P3a helix from both aptamers. Mutation of these residues altered both the cooperativity and binding affinity of the riboswitch. The data support a model in which the P1 helix of the first aptamer participates in a tertiary interaction important for cooperativity, while nucleotides in the P1 helix of the second aptamer interface with the expression platform. These data have direct analogy to well-characterized mutations in hemoglobin, which provides a framework for considering cooperativity in this RNA-based system.     

The molecular basis of cooperativity in protein folding. Thermodynamic dissection of interdomain interactions in phosphoglycerate kinase.

In the presence of guanidine hydrochloride, phosphoglycerate kinase from yeast can be reversibly denatured by either heating or cooling the protein solution above or below room temperature [Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278]. The heat denaturation of PGK is characterized by the presence of a single peak in the excess heat capacity function obtained by differential scanning calorimetry. The transition curve approaches the two-state mechanism, indicating that the two domains of the molecule display strong cooperative interactions and that partially folded intermediates are not largely populated during the transition. On the contrary, the cold denaturation is characterized by the presence of two peaks in the heat capacity function. Analysis of the data indicates that at low temperatures the two domains behave independently of each other. The crystallographic structure of PGK has been used to identify the nature of the interactions between the two domains. These interactions involve primarily the apposition of two hydrophobic surfaces of approximately 480 A2 and nine hydrogen bonds. This information, in conjunction with experimental thermodynamic values for hydrophobic, hydrogen bonding interactions and statistical thermodynamic analysis, has been used to quantitatively account for the folding/unfolding behavior of PGK. It is shown that this type of analysis accurately predicts the cooperative behavior of the folding/unfolding transition and its dependence on GuHCl concentration.     

Structural basis of negative cooperativity in transthyretin

A comparison of the AC and BD binding sites of transthyretin (TTR) was made in terms of the interatomic distances between the Ca atoms of equivalent amino acids, measured across the tetramer channel in each binding site. The comparison of the channel diameter for apo TTR from different sources revealed that in the unliganded transthyretin tetramers the distances between the A, D and H beta-strands are consistently larger, while the distances between the G beta-strands are smaller in one site than in the other. These differences might be described to have a 'wave' character. An analogous analysis performed for transthyretin complexes reveals that the shape of the plot is similar, although the amplitudes of the changes are smaller. The analysis leads us to a model of the changes in the binding sites caused by ligand binding. The sequence of events includes ligand binding in the first site, followed by a slight collapse of this site and concomitant opening of the second site, binding of the second molecule and collapse of the second site. The following opening of the first, already occupied site upon ligand binding in the second site is smaller because of the bridging interactions already formed by the first ligand. This explains the negative cooperativity (NC) effect observed for many ligands in transthyretin.     

Structural basis of cooperativity in human UDP-glucose dehydrogenase

Background

UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.

Methodology

Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.

Conclusion

In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD⁺ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD⁺ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.     

The structural basis of negative cooperativity: receptors and enzymes

Crystal structures of the negatively cooperative aspartate receptor caught at intermediate stages in the binding process help to elucidate structural factors involved in ligand binding. The frequency of occurrence of negatively cooperative proteins suggests that sequential changes in binding patterns will be extensive in positively cooperative as well as in negatively cooperative and no cooperativity proteins.     

Structural basis of affinity maturation and intramolecular cooperativity in a protein-protein interaction

Although protein-protein interactions are involved in nearly all cellular processes, general rules for describing affinity and selectivity in protein-protein complexes are lacking, primarily because correlations between changes in protein structure and binding energetics have not been well determined. Here, we establish the structural basis of affinity maturation for a protein-protein interaction system that we had previously characterized energetically. This model system exhibits a 1500-fold affinity increase. Also, its affinity maturation is restricted by negative intramolecular cooperativity. With three complex and six unliganded variant X-ray crystal structures, we provide molecular snapshots of protein interface remodeling events that span the breadth of the affinity maturation process and present a comprehensive structural view of affinity maturation. Correlating crystallographically observed structural changes with measured energetic changes reveals molecular bases for affinity maturation, intramolecular cooperativity, and context-dependent binding.     

Osmotic pressure: Thermodynamic basis and units of measurement

The theoretical basis and practical implications of osmotic pressure measurement are briefly discussed. Stress is laid on interconversions of various tabulated and practically used units and on practical examples of osmotic pressure calculation and determination, with special reference to possible pitfalls in osmotic pressure assessment and adjustment. Part I of a series of overviews on osmotic pressure and osmosis.     

Thermodynamic model of cooperativity in a dimeric protein: unique and independent parameters formulation.

A model of the cooperative interaction of ligand binding to a dimeric protein is presented based upon the unique and independent parameters (UIP) thermodynamic formulation (Gutheil and McKenna, Biophys. Chem. 45 (1992) 171-179). The analysis is developed from an initial model which includes coupled conformational and ligand binding equilibria. This completely general model is then restricted to focus on conformationally mediated cooperative interactions between the ligands and the expressions for the apparent ligand binding constant and the apparent ligand-ligand interaction constant are derived. The conditions under which there is no cooperative interaction between the ligands are found as roots to a polynomial equation. Consideration of the distribution of species among the various conformational states in this general model leads to a set of inequalities which can be represented as a two dimensional plot of boundaries. By superimposing a contour plot of the value of the apparent ligand-ligand interaction constant over the plot of boundaries a complete graphical representation of this system is achieved similar to a phase diagram. It is found that the parameter space homologous to Koshland-Nemethy-Filmer type of model is most consistent with both positive and negative cooperativity in this model. The maximal amount of positive and negative cooperativity are found to be simple functions of Kc, the equilibrium constant associated with the change of a subunit and ligand from the unligated to ligated conformation. It is shown that under certain limiting conditions the apparent allosteric interaction between ligands is equal to the conformational interaction between subunits. The methods presented are generally applicable to the theoretical analysis of thermodynamic interactions in complex systems.     

Structural basis of folding cooperativity in model proteins: insights from a microcanonical perspective

Two-state cooperativity is an important characteristic in protein folding. It is defined by a depletion of states that lie energetically between folded and unfolded conformations. There are different ways to test for two-state cooperativity; however, most of these approaches probe indirect proxies of this depletion. Generalized-ensemble computer simulations allow us to unambiguously identify this transition by a microcanonical analysis on the basis of the density of states. Here, we present a detailed characterization of several helical peptides obtained by coarse-grained simulations. The level of resolution of the coarse-grained model allowed to study realistic structures ranging from small α-helices to a de novo three-helix bundle without biasing the force field toward the native state of the protein. By linking thermodynamic and structural features, we are able to show that whereas short α-helices exhibit two-state cooperativity, the type of transition changes for longer chain lengths because the chain forms multiple helix nucleation sites, stabilizing a significant population of intermediate states. The helix bundle exhibits signs of two-state cooperativity owing to favorable helix-helix interactions, as predicted from theoretical models. A detailed analysis of secondary and tertiary structure formation fits well into the framework of several folding mechanisms and confirms features that up to now have been observed only in lattice models.     

Thermodynamic basis for sequence-specific recognition of ssDNA by an autoantibody

11F8 is a sequence-specific DNA binding monoclonal autoantibody previously isolated from an autoimmune lupus-prone mouse [Stevens, S. Y., and Glick, G. D. (1999) Biochemistry 38, 560-568]. This antibody, like many other lupus anti-DNAs, localizes to kidney tissue and eventually leads to renal damage through a process that first involves the binding of DNA antigens. A series of experiments were conducted to investigate the thermodynamic and structural basis by which this antibody discriminates between specific, noncognate, and nonspecific sequences. Sequence-specific binding occurs with a minimal dependence on the polyelectrolyte effect along with a favorable binding enthalpy reflecting the presence of base stacking and contacts to DNA bases. This favorable binding enthalpy apparently is derived from desolvation at the binding interface and is consistent with recent models of the nonclassical hydrophobic effect. Noncognate recognition is also driven by the nonclassical hydrophobic effect, but is accompanied by highly unfavorable entropies that are responsible for reduced affinity relative to the high-affinity consensus sequence. Nonspecific recognition is driven completely by the polyelectrolyte effect involving extensive electrostatic interactions with the phosphate backbone. Collectively, the data demonstrate the ability of 11F8 to adapt its mode of binding to the available DNA surface and provide a thermodynamic model for sequence-specific recognition of single-stranded DNA. The salient features of this model employ the paradigms invoked to explain protein.dsDNA, protein.RNA, and antibody.antigen binding.     

Thermodynamic and structural basis for transition-state stabilization in antibody-catalyzed hydrolysis

Catalytic antibodies 6D9 and 9C10, which were induced by immunization with a haptenic transition-state analog (TSA), catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. These antibodies stabilize the transition state to catalyze the hydrolysis reaction, strictly according to the theoretical relationship: for 6D9, k(cat)/k(uncat)=895 and K(S)/K(TSA)=900, and for 9C10, k(cat)/k(uncat)=56 and K(S)/K(TSA)=60. To elucidate the molecular basis of the antibody-catalyzed reaction, the crystal structure of 6D9 was determined, and the binding thermodynamics of 6D9 and 9C10 with both the substrate and the TSA were analyzed using isothermal titration calorimetry. The crystal structure of the unliganded 6D9 Fab was determined at 2.25 A resolution and compared with that of the TSA-liganded 6D9 Fab reported previously, showing that the TSA is bound into the hydrophobic pocket of the antigen-combining site in an "induced fit" manner, especially at the L1 and H3 CDR loops. Thermodynamic analyses showed that 6D9 binds the substrate of the TSA with a positive DeltaS, differing from general thermodynamic characteristics of antigen-antibody interactions. This positive DeltaS could be due to the hydrophobic interactions between 6D9 and the substrate or the TSA mediated by Trp H100i. The difference in DeltaG between substrate and TSA-binding to 6D9 was larger than that to 9C10, which is in good correlation with the larger k(cat) value of 6D9. Interestingly, the DeltaDeltaG was mainly because of the DeltaDeltaH. The correlation between k(cat) and DeltaDeltaH is suggestive of "enthalpic strain" leading to destabilization of antibody-substrate complexes. Together with X-ray structural analyses, the thermodynamic analyses suggest that upon binding the substrate, the antibody alters the conformation of the ester moiety in the substrate from the planar Z form to a thermodynamically unstable twisted conformation, followed by conversion into the transition state. Enthalpic strain also contributes to the transition-state stabilization by destabilizing the ground state, and its degree is much larger for the more efficient catalytic antibody, 6D9.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

Structural basis of binding of fluorescent, site-specific dansylated amino acids to human serum albumin

Human serum albumin (HSA) has two primary binding sites for drug molecules. These sites selectively bind different dansylated amino acid compounds, which-due to their intrinsic fluorescence-have long been used as specific markers for the drug pockets on HSA. We present here the co-crystal structures of HSA in complex with six dansylated amino acids that are specific for either drug site 1 (dansyl-l-asparagine, dansyl-l-arginine, dansyl-l-glutamate) or drug site 2 (dansyl-l-norvaline, dansyl-l-phenylalanine, dansyl-l-sarcosine). Our results explain the structural basis of the site-specificity of different dansylated amino acids. They also show that fatty acid binding has only a modest effect on binding of dansylated amino acids to drug site 1 and identify the location of secondary binding sites.