Maria Håkansson

Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein−ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. 15N and 2H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein−carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

Crystal Structure of a Superantigen Bound to MHC Class II Displays Zinc and Peptide Dependence

The three‐dimensional structure of a bacterial superantigen, Staphylococcus aureus enterotoxin H (SEH), bound to human major histocompatibility complex (MHC) class II (HLA‐DR1) has been determined by X‐ray crystallography to 2.6 Å resolution (1HXY). The superantigen binds on top of HLA‐DR1 in a completely different way from earlier co‐crystallized superantigens from S.aureus. SEH interacts with high affinity through a zinc ion with the β1 chain of HLA‐DR1 and also with the peptide presented by HLA‐DR1. The structure suggests that all superantigens interacting with MHC class II in a zinc‐dependent manner present the superantigen in a common way. This suggests a new model for ternary complex formation with the T‐cell receptor (TCR), in which a contact between the TCR and the MHC class II is unlikely.

Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.

The bacterial ω‐transaminase from Chromobacterium violaceum (Cv‐ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5′‐phosphate (PLP). Here, we present four crystal structures of Cv‐ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo‐Cv‐ωTA structure reveals unique ‘relaxed’ conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered ‘roof ’ over the PLP‐binding site, whereas in the other apo form and the holo form the ‘roof’ is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the ‘roof’ structure may be associated with substrate binding in Cv‐ωTA and some other transaminases.

The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate–protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 Å at 100 K, 1.25 Å at 298 K) and in complex with lactose (0.86 Å) or glycerol (0.9 Å). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.

An extended hudrophobic core induces EF‐hand swapping

The structure of calbindin D9k with two substitutions was determined by X‐ray crystallography at 1.8‐Å resolution. Unlike wild‐type calbindin D9k, which is a monomeric protein with two EF‐hands, the structure of the mutated calbindin D9k reveals an intertwined dimer. In the dimer, two EF‐hands of the monomers have exchanged places, and thus a 3D domain‐swapped dimer has been formed. EF‐hand I of molecule A is packed toward EF‐hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF‐hands, promotes the conversion of monomers to 3D domain‐swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D9k. Once formed, the calbindin D9k dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D9k dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.

Protein reconstitution and 3D domain swapping.

The native structures of proteins are governed by a large number of non-covalent interactions yielding a high specificity for the native packing of structural elements. This allows for the reconstitution of proteins from disconnected polypeptide fragments. The specificity for the native arrangement also enables interchange of structural elements with another identical protein chain resulting in dimers with swapped segments. Proteins are not static structures, but open up repetitively on a timescale of minutes to years depending on the identity of the protein and solution conditions. The open protein may self-close and return to the native state, or it may close with another polypeptide chain leading to 3D domain swapping. The term describes two or more protein molecules swapping identical domains or smaller secondary structure elements. The non-covalent intra-molecular interactions between domains in the monomer are thus broken and restored in the oligomer by identical inter-molecular contacts. This review will discuss 3D domain swapping in relation to protein reconstitution and fibril formation. Examples of reconstituted and domain-swapped proteins will be given. The physiological benefits of 3D domain swapping will be discussed, as well as its role in the evolution of proteins and pathology.

Crystal Structure of N-Glycosylated Human Glypican-1 Core Protein: Structure of Two Loops Evolutionarily Conserved in Vertebrate Glypican-1.

Background: The glypican family of cell-surface proteoglycans regulates growth factor signaling during development through their core proteins and heparan sulfate chains. Results: The crystal structure of N-glycosylated human glypican-1 is described. Conclusion: The structure reveals the complete disulfide bond arrangement for the conserved Cys residues in glypicans. Significance: Increased structural knowledge of glypicans will help elucidate their important functions in shaping animal development. Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.

Structure-function analysis of heterodimer formation, oligomerization, and receptor binding of the Staphylococcus aureus bi-component toxin LukGH.

Background:LukGH is a member of the family of two-component bacterial toxins of Staphylococcus aureus that lyse human phagocytic cells. Results:The crystal structure of LukGH and mutagenesis revealed the molecular basis for heterodimer formation in solution. Conclusion:LukGH differs from other two-component leukocidins that interact only upon cell contact. Significance:These data might assist with development of therapeutics that counteract Staphylococcus aureus pathogenesis. The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30–40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Å resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.

Supplementation by thylakoids to a high carbohydrate meal decreases feelings of hunger, elevates CCK levels and prevents postprandial hypoglycaemia in overweight women

Thylakoids are chlorophyll-containing membranes in chloroplasts that have been isolated from green leaves. It has been previously shown that thylakoids supplemented with a high-fat meal can affect cholecystokinin (CCK), ghrelin, insulin and blood lipids in humans, and can act to suppress food intake and prevent body weight gain in rodents. This study investigates the addition of thylakoids to a high carbohydrate meal and its effects upon hunger motivation and fullness, and the levels of glucose, insulin, CCK, ghrelin and tumour necrosis factor (TNF)-alpha in overweight women. Twenty moderately overweight female subjects received test meals on three different occasions; two thylakoid enriched and one control, separated by 1 week. The test meals consisted of a high carbohydrate Swedish breakfast, with or without addition of thylakoids. Blood samples and VAS-questionnaires were evaluated over a 4-h period. Addition of thylakoids suppressed hunger motivation and increased secretion of CCK from 180 min, and prevented postprandial hypoglycaemia from 90 min following food intake. These effects indicate that thylakoids may intensify signals of satiety. This study therefore suggests that the dietary addition of thylakoids could aid efforts to reduce food intake and prevent compensational eating later in the day, which may help to reduce body weight over time.

Structural Basis for Carbohydrate-Binding Specificity--A Comparative Assessment of Two Engineered Carbohydrate-Binding Modules.

Detection, immobilization and purification of carbohydrates can be done using molecular probes that specifically bind to targeted carbohydrate epitopes. Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that can be engineered to bind and detect specifically a number of carbohydrates. Design and engineering of CBMs have benefited greatly from structural studies that have helped us to decipher the basis for specificity in carbohydrate-protein interactions. However, more studies are needed to predict which modifications in a CBM would generate probes with predetermined binding properties. In this report, we present the crystal structures of two highly related engineered CBMs with different binding specificity profiles: X-2, which is specific for xylans and the L110F mutant of X-2, which binds xyloglucans and β-glucans in addition to xylans. The structures of the modules were solved both in the apo form and complexed with oligomers of xylose, as well as with an oligomer of glucose in the case of X-2 L110F. The mutation, leucine to phenylalanine, converting the specific module into a cross-reactive one, introduces a crucial hydrogen-π interaction that allows the mutant to retain glucan-based ligands. The cross-reactivity of X-2 L110F is furthermore made possible by the plasticity of the protein, in particular, of residue R142, which permits accommodation of an extra hydroxymethyl group present in cellopentaose and not xylopentaose. Altogether, this study shows, in structural detail, altered protein-carbohydrate interactions that have high impact on the binding properties of a carbohydrate probe but are introduced through simple mutagenesis.