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 ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). 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 beta-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

Galectin-3 alters the lateral mobility and clustering of beta 1-integrin receptors

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the alpha 5 beta 1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased beta 1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins

A study of aromatic hydrogen bonds of peptides with aromatic amino acid side-chains

184-187The importance of hydrogen bonding studies lies in their structural, biological and medicinal applications. Non-conventional hydrogen bonds are weak, but are found to play an important role in biological molecules. In view of their importance a study of the aromatic hydrogen bonds in peptides with aromatic, amino acid side chains was carried out. The results indicate a reasonable probability for their occurrence, thereby enumerating their distinct features

Molecular dynamics of a far positioned SOD1 mutant V14M reveals pathogenic misfolding behavior

<p>Human superoxide dismutase (Cu/Zn SOD1) is a homodimeric enzyme. Mutations in Cu/Zn SOD1 causes a familial form of amyotrophic lateral sclerosis (fALS), and aggregation of mutant SOD1 has been proposed to play a role in neurodegeneration. Though a majority of the mutations are point substitutions, there are a few changes that result in amino acid deletions or truncations of the polypeptide. These pathogenic mutations are scattered throughout the three-dimensional structure of the dimeric enzyme, which creates a puzzling pattern to investigate the molecular determinants of fALS. The most common hypothesis proposed that the misfolding of SOD1 mutants are primarily triggered by decreased affinity for metal ions. However, this hypothesis is challenging, as a significant number of disease-causing mutations are located far away from the metal-binding site and dimer interface. So in the present study, we have investigated the influence of such a far positioned pathogenic mutation, V14M, in altering the stability and folding of the Cu/Zn SOD1. Though the location of Val14 is far positioned, it has a vital role in the stability of SOD1 by preserving its hydrophobic cluster at one end of the β barrel domain. We have performed MD simulations of the V14M mutant for 80 ns timescale. The results reveal the fact that irrespective of its location, V14M mutation triggers a conformational change that is more similar to that of the metal-deficient holo form and could resemble an intermediate state in the folding reaction which results in protein misfolding and aggregation.</p

Folding Catalysis by Transient Coordination of Zn2+ to the Cu Ligands of the ALS-Associated Enzyme Cu/Zn Superoxide Dismutase 1

How coordination of metal ions modulates protein structures is not only important for elucidating biological function but has also emerged as a key determinant in protein turnover and protein-misfolding diseases. In this study, we show that the coordination of Zn2+ to the ALS-associated enzyme Cu/Zn superoxide dismutase (SOD1) is directly controlled by the protein's folding pathway. Zn2+ first catalyzes the folding reaction by coordinating transiently to the Cu ligands of SOD1, which are all contained within the folding nucleus. Then, after the global folding transition has commenced, the Zn2+ ion transfers to the higher affinity Zn site, which structures only very late in the folding process. Here it remains dynamically coordinated with an off rate of similar to 10(-5) s(-1). This relatively rapid equilibration of metals in and out of the SOD1 structure provides a simple explanation for how the exceptionally long lifetime, >100 years, of holoSOD1 is still compatible with cellular turnover: if a dissociated Zn2+ ion is prevented from rebinding to the SOD1 structure then the lifetime of the protein is reduced to a just a few hours

Systematic study on crystal-contact engineering of diphthine synthase: influence of mutations at crystal-packing regions on X-ray diffraction quality

It is well known that protein crystallizability can be influenced by site-directed mutagenesis of residues on the molecular surface of proteins, indicating that the intermolecular interactions in crystal-packing regions may play a crucial role in the structural regularity at atomic resolution of protein crystals. Here, a systematic examination was made of the improvement in the diffraction resolution of protein crystals on introducing a single mutation of a crystal-packing residue in order to provide more favourable packing interactions, using diphthine synthase from Pyrococcus horikoshii OT3 as a model system. All of a total of 21 designed mutants at 13 different crystal-packing residues yielded almost isomorphous crystals from the same crystallization conditions as those used for the wild-type crystals, which diffracted X-rays to 2.1 angstrom resolution. Of the 21 mutants, eight provided crystals with an improved resolution of 1.8 angstrom or better. Thus, it has been clarified that crystal quality can be improved by introducing a suitable single mutation of a crystal-packing residue. In the improved crystals, more intimate crystal-packing interactions than those in the wild-type crystal are observed. Notably, the mutants K49R and T146R yielded crystals with outstandingly improved resolutions of 1.5 and 1.6 angstrom, respectively, in which a large-scale rearrangement of packing interactions was unexpectedly observed despite the retention of the same isomorphous crystal form. In contrast, the mutants that provided results that were in good agreement with the designed putative structures tended to achieve only moderate improvements in resolution of up to 1.75 angstrom. These results suggest a difficulty in the rational prediction of highly effective mutations in crystal engineering