K. Saraboji

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.

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 Zn(2+) to the ALS-associated enzyme Cu/Zn superoxide dismutase (SOD1) is directly controlled by the proteins folding pathway. Zn(2+) 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 Zn(2+) 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 ∼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 Zn(2+) ion is prevented from rebinding to the SOD1 structure then the lifetime of the protein is reduced to a just a few hours.

Hydrophobic environment is a key factor for the stability of thermophilic proteins

The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter‐residue interactions, ion‐pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic–mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three‐dimensional structures of elongation factor‐Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the importance of hydrophobicity as the dominating characteristic in the stability of thermophilic proteins, and we anticipate this will be useful in our attempts to engineering thermostable proteins.

Global structural motions from the strain of a single hydrogen bond

The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the β-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120–H43–T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?

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 A resolution. Of the 21 mutants, eight provided crystals with an improved resolution of 1.8 A 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 A, 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 A. These results suggest a difficulty in the rational prediction of highly effective mutations in crystal engineering.

Contribution of main chain and side chain atoms and their locations to the stability of thermophilic proteins

Proteins belonging to the same class, having similar structures thus performing the same function are known to have different thermal stabilities depending on the source- thermophile or mesophile. The variation in thermo-stability has not been attributed to any unified factor yet and understanding this phenomenon is critically needed in several areas, particularly in protein engineering to design stable variants of the proteins. Toward this motive, the present study focuses on the sequence and structural investigation of a dataset of 373 pairs of proteins; a thermophilic protein and its mesophilic structural analog in each pair, from the perspectives of hydrophobic free energy, hydrogen bonds, physico-chemical properties of amino acids and residue-residue contacts. Our results showed that the hydrophobic free energy due to carbon, charged nitrogen and charged oxygen atoms was stronger in 65% of thermophilic proteins. The number of hydrogen bonds which bridges the buried and exposed regions of proteins was also greater in case of thermophiles. Amino acids of extended shape, volume and molecular weight along with more medium and long range contacts were observed in many of the thermophilic proteins. These results highlight the preference of thermophiles toward the amino acids with larger side chain and charged to make up greater free energy, better packing of residues and increase the overall compactness.

Perdeuteration, crystallization, data collection and comparison of five neutron diffraction data sets of complexes of human galectin-3C

Perdeuteration, purification and the growth of large crystals of the carbohydrate-recognition domain of galectin-3C are described. Five neutron diffraction data sets have been collected at four neutron sources; these are compared and two are merged.

Conformational Aspects and Interaction Studies of Heterocyclic Drugs

Drug discoveries require the iterative synthesis—along with structural studies— of numerous individual analogues of biologically and medicinally active compounds. Over half of all known compounds and a large number of pharmaceutical products are heterocyclic in nature. The pharmacological activity of drugs depends mainly on interaction with their biological targets, which have a complex three-dimensional structure, and molecular recognition is guided by the nature of the intermolecular interactions. Furthermore, the drug’s polymorphic nature also adversely affects its abilities. In order to address these factors, the stereochemical analysis of various piperidine and azepine derivatives, weak π-interaction analysis of isoxazole, imidazole, indole, quinoline and triazole and polymorphic analysis of two commercial drugs, valdecoxib and sildenafil citrate were carried out. Only the crystal structures were used for these analyses, of which the piperidine and azepine derivatives, valdecoxib and sildenafil citrate were solved by our group. To understand the structure-activity relationship, the results of these studies were correlated with the crystal structure of their respective drug molecules that are found in complex with the receptors. Stereochemical analysis showed that the ring conformation and orientation of the substituents correlate well with the active conformation of the drug. The π-systems prefer to form an offset stacking π. . .π interaction geometry similar to the phenylalanine–phenylalanine interactions in proteins. In the polymorphic analysis one of the crystal conformations of valdecoxib proved to have better interaction with its receptor indicating higher activity.

CRYSTAL STRUCTURE AND CONFORMATION OF TWO SIMILAR PIPERIDONES

N-Chloroacetyl-3,5-dimethyl-2,6-diphenylpiperidin-4-one(CADMPO), C21H22ClNO2, F.W=355.85, monoclinic, P21, a=8.2082(10) Å, b=10.4889(10) Å, c=10.6175(10) Å, β=91.833(10)°, V=913.73(17) Å3, Z=2, Dcalc=1.293 Mg/m3, μ=1.953 mm−1, F000=376, CuKα=1.5418 Å, final R1 and wR2 are 0.0399 and 0.0911, respectively N-Chloroacetyl-3-ethyl-2,6-diphenylpiperidin-4-one (CAEPO), C21H22ClNO2, F.W=355.85, monoclinic, P21/n, a=10.3626(6) Å, b=8.5702(5) Å, c=21.6930(10) Å, β=92.25(1)°, V=1925.06(18) Å3, Z=8, Dcalc=1.228 Mg/m3, μ=0.211 mm−1, F000=752, MoKα=0.71073 Å, final R1 and wR2 are 0.0623 and 0.1397, respectively. Crystal structure studies of these two 4-piperidones show that the piperidones adopt twist-boat conformation. The C–H…O type of intermolecular interactions play a role in stabilizing the molecules in the unit cell in addition to van der Waals forces.

CRYSTAL STRUCTURE AND CONFORMATION OF A PAIR OF PIPERIDINE DERIVATIVES

N-Morpholinoacetyl-3-methyl-2,6-diphenylpiperidin-4-one(MCAMPO), C 24 H 28 N 2 O 3 , F.W = 392.48, monoclinic, P2 1 /c, α = 9.5714(10) A, b = 19.7588(10)A, c = 11.3961(10)A, β = 94.383(10)°, V = 2148.9(3)A 3 , Z = 4, D calc = 1.213 Mg/m 3 , μ = 0.639 mm -1 , F 000 = 840, CuKα = 1.5418 A, final R1 and wR2 are 0.0509 and 0.1352, respectively. N-Morpholinoacetyl-3-isopropyl-2,6-diphenylpiperidin-4-one(MCAIPO), C 26 H 32 N 2 O 3 , F.W = 420.54, orthorhombic, P2 1 2 1 2 1 , α = 9.0053(2) A, b = 12.1942(10) A c = 21.1742(2) A, V = 2325.19(6)A 3 , Z = 4, D calc = 1.201 Mg/m 3 , μ = 0.078 mm -1 , F 000 = 904, MoKα = 0.71073A final R1 and wR2 are 0.0595 and 0.1151, respectively. The heterocyclic rings of the two ketopiperidines exhibit twist-boat and chair conformations, respectively. The morpholine ring poses an acceptor nitrogen atom for C-H...N interactions in both the structures.