Crystal structure of beta1,4-galactosyltransferase complex with UDP-Gal reveals an oligosaccharide acceptor binding site.

The crystal structure of the catalytic domain of bovine beta1,4-galactosyltransferase (Gal-T1) co-crystallized with UDP-Gal and MnCl(2) has been solved at 2.8 A resolution. The structure not only identifies galactose, the donor sugar binding site in Gal-T1, but also reveals an oligosaccharide acceptor binding site. The galactose moiety of UDP-Gal is found deep inside the catalytic pocket, interacting with Asp252, Gly292, Gly315, Glu317 and Asp318 residues. Compared to the native crystal structure reported earlier, the present UDP-Gal bound structure exhibits a large conformational change in residues 345-365 and a change in the side-chain orientation of Trp314. Thus, the binding of UDP-Gal induces a conformational change in Gal-T1, which not only creates the acceptor binding pocket for N-acetylglucosamine (GlcNAc) but also establishes the binding site for an extended sugar acceptor. The presence of a binding site that accommodates an extended sugar offers an explanation for the observation that an oligosaccharide with GlcNAc at the non-reducing end serves as a better acceptor than the monosaccharide, GlcNAc. Modeling studies using oligosaccharide acceptors indicate that a pentasaccharide, such as N-glycans with GlcNAc at their non-reducing ends, fits the site best. A sequence comparison of the human Gal-T family members indicates that although the binding site for the GlcNAc residue is highly conserved, the site that binds the extended sugar exhibits large variations. This is an indication that different Gal-T family members prefer different types of glycan acceptors with GlcNAc at their non-reducing ends.

Molecular dynamics simulations of hybrid and complex type oligosaccharides.

Conformational preferences of hybrid (GlcNAc1Man5GlcNAc2) and complex (GlcNAc1Man3GlcNAc2; GlcNAc2Man3GlcNAc2) type asparagine-linked oligosaccharides and the corresponding bisected oligosaccharides have been studied by molecular dynamics simulations for 2.5 ns. The fluctuations of the core Man-alpha 1,3-Man fragment are restricted to a region around (-30 degrees, -30 degrees) due to a 'face-to-face' arrangement of bisecting GlcNAc and the beta 1,2-GlcNAc on the 1,3-arm. However, conformations where such a 'face-to-face' arrangement is disrupted are also accessed occasionally. The orientation of the 1,6-arm is affected not only by changes in chi, but also by changes in phi and psi around the core Man-alpha 1,6-Man linkage. The conformation around the core Man-alpha 1,6-Man linkage is different in the hybrid and the two complex types suggesting that the preferred values of phi, psi, and chi are affected by the addition or deletion of saccharides to the alpha 1,6-linked mannose. The conformational data are in agreement with the available experimental studies and also explain the branch specificity of galactosyltransferases.

Functional domains of bovine beta-1,4 galactosyltransferase.

A number of N- and C-terminal deletion and point mutants of bovine beta-1,4 galactosyltransferase (beta-1,4GT) were expressed in E. coli to determine the binding regions of the enzyme that interact with N-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1-129, do not show any significant difference in the apparent Kms towards NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130-402 of bovine beta-1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15 mM NAG. The C-terminus fragment GT-257UDP, containing residues 258-402 of beta-1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of beta-1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+.

Molecular dynamics simulations of oligosaccharides and their conformation in the crystal structure of lectin-carbohydrate complex: importance of the torsion angle psi for the orientation of alpha 1,6-arm.

The conformation of the heptasaccharide Man-alpha 1,6-(Man-alpha 1,3)(Xyl-beta 1,2)-Man-beta 1,4-GlcNAc2-beta 1,4-(L- Fuc-alpha 1,3)-GlcNAc1, the carbohydrate moiety of Erythrina corallodendron lectin (EcorL), the hexasaccharide Man-alpha 1,6-(Man-alpha 1,3) (GlcNAc-beta 1,4)-Man-beta 1,4-GlcNAc-beta 1,4-GlcNAc and their disaccharide fragments have been studied by molecular dynamics (MD) simulations for 1000 ps with different initial conformations. In the isolated heptasaccharide, the most frequently accessed conformation during MD has a psi value of 180 degrees around Man-alpha 1,6-Man linkage. This conformation is stabilized by the formation of a hydrogen bond between the carbonyl oxygen of GlcNAc2 with the O3/O4 hydroxyls of the alpha 1,6-linked mannose residue. The conformation of the heptasaccharide found in the crystal structure of the EcorL-lactose complex (Shaanan et al., Science, 254, 862, 1991), that has a psi value of approximately 76 degrees around Man-alpha 1,6-Man linkage, is accessed, although less frequently, during MD of the isolated oligosaccharide. The phi, psi, chi = 58 degrees, -134 degrees, -60 degrees conformation around Man-alpha 1,6-Man fragment observed in the crystal structure of the Lathyrus ochrus lectin complexed with a biantennary octasaccharide (Table I in Homans, S.W., Glycobiology, 3, 551, 1993) has also been accessed in the present MD simulations. These psi values for the alpha 1,6-linkage, which are observed in the protein-carbohydrate crystal structures and are accessed in the MD simulations, though occasionally, have not been predicted from NMR studies. Furthermore, these different values of psi lead to significantly different orientations of the alpha 1,6-arm for the same value of chi. This contrasts with the earlier predictions that only different values of chi can bring about significant changes in the orientation of the alpha 1,6-arm. The MD simulations also show that the effects of bisecting GlcNAc or beta 1,2-xylose are very similar on the alpha 1,3-arm and slightly different on the alpha 1,6-arm.

Molecular dynamics simulations of high-mannose oligosaccharides.

Conformations of several high-mannose-type oligosaccharides that are generated during the biosynthetic degradation of Man9GlcNAc2 to Man5GlcNAc2 have been studied by molecular dynamics (MD). Simulations were performed on NCI-FCRDC's Cray Y-MP 8D/8128 supercomputer using Biosym's CVFF force field for 1000 ps with different initial conformations. The conformations of the two alpha 1,3- and the two alpha 1,6-linkages in each oligomannose were different, suggesting that deriving oligosaccharide conformations based on the conformational preferences of the constituent disaccharide fragments will not always yield correct results. Unlike other oligomannoses, Man9GlcNAc2 appears to take more than one distinct conformation around the core alpha 1,6-linkage. These various conformations may play an important role in determining the processing pathways. Using the data on the preferred conformations of these oligomannoses and the available experimental results, possible pathways for processing Man9GlcNAc2 to Man5GlcNAc2 by alpha 1,2-linkage-specific mannosidases have been proposed. Conformational analysis of Man5GlcNAc2 indicates that the addition of beta 1,2-GlcNAc to the alpha 1,3-linked core mannose, besides serving as a prerequisite for mannosidase II action as suggested earlier, may also prevent the removal of alpha 1,3-mannose. The MD simulations also suggest that the processing of the precursor oligosaccharide during Asn-linked complex and hybrid glycan biosynthesis proceeds in a well-defined pathway involving more than one alpha 1,2-linkage-specific mannosidase. Knowledge of the conformation of the processing intermediates obtained from the present study can be used to design highly specific substrate analogues to inhibit a particular mannosidase, thereby blocking one processing pathway without interfering with the others.

Molecular dynamics simulations of asialoglycoprotein receptor ligands.

Several recent studies have implicated carbohydrates in cell adhesion, inflammation, clearance of glycoproteins from blood circulation, embryonic development, and metastasis among others. Understanding the conformation of these carbohydrate recognition elements and their interaction at the molecular level is essential for the design of oligosaccharide inhibitors/drugs. Given the difficulty in solving carbohydrate structures by X-ray crystallography and since NMR experiments give only time-averaged conformation, molecular dynamics simulations are well suited to determine all the accessible conformations of oligosaccharides. Present communication reports the simulation of some of the oligosaccharide ligands of asialoglycoprotein receptor for 1 ns using Biosym's Insight II molecular modeling package on NCI-FCRDC's Y-MP 8D/8128 supercomputer. Results obtained from these simulations, in addition to explaining the observed differences in the binding affinities of these ligands to the asialoglycoprotein receptor, have led to a modified model for the recognition of the oligosaccharides by the receptor. Accordingly, only the two terminal galactose residues on the 1,3-arm of the triantennary oligosaccharide (GlcNAc2Man3 core of the N-linked oligosaccharides with N-acetyllactosamine in beta 1,2- and beta 1,4-linkages on the 1,3-linked core mannose) are primarily required for recognition, and the terminal galactose on the 1,6-arm (N-acetyllactosamine in beta 1,2-linkage on the 1,6-linked core mannose) provides additional binding energy. It has been shown that the oligosaccharides studied here have significant flexibility and the flexibility is more around the 1,3-linkage than the 1,6-linkage. The need for simulation for longer periods and with multiple initial conformations is also discussed in the present report.

Computer modelling studies of ribonuclease A-pyrimidine nucleotide complexes.

Different modes of binding of pyrimidine monophosphates 2'-UMP, 3'-UMP, 2'-CMP and 3'-CMP to ribonuclease (RNase) A are studied by energy minimization in torsion angle and subsequently in Cartesian coordinate space. The results are analysed in the light of primary binding sites. The hydrogen bonding pattern brings out roles for amino acids such as Asn44 and Ser123 apart from the well known active site residues viz., His12,Lys41,Thr45 and His119. Amino acid segments 43-45 and 119-121 seem to be guiding the ligand binding by forming a pocket. Many of the active site charged residues display considerable movement upon nucleotide binding.

Expression of deletion constructs of bovine beta-1,4-galactosyltransferase in Escherichia coli: importance of Cys134 for its activity.

Bovine beta-1,4-galactosyltransferase (beta-1,4-GT; EC 2.4.1.90) belongs to the glycosyltransferase family and as such shares a general topology: an N-terminal cytoplasmic tail, a signal anchor followed by a stem region and a catalytic domain at the C-terminal end of the protein. cDNA constructs of the N-terminal deleted forms of beta-1,4-GT were prepared in pGEX-2T vector and expressed in E. coli as glutathione-S-transferase (GST) fusion proteins. Recombinant proteins accumulated within inclusion bodies as insoluble aggregates that were solubilized in 5 M guanidine HCl and required an 'oxido-shuffling' reagent for regeneration of the enzyme activity. The recombinant beta-1,4-GT, devoid of the GST domain, has 30-85% of the sp. act. of bovine milk beta-1,4-GT with apparent Kms for N-acetylglucosamine and UDP-galactose similar to those of milk enzyme. Deletion analyses show that both beta-1,4-GT and lactose synthetase activities remain intact even in the absence of the first 129 residues (pGT-d129). The activities are lost when either deletions extend up to residue 142 (pGT-d142) or Cys134 is mutated to Ser (pGT-d129C134S). These results suggest that the formation of a disulfide bond involving Cys134 holds the protein in a conformation that is required for enzymatic activity.

Mutational analysis of the Golgi retention signal of bovine beta-1,4-galactosyltransferase.

To examine the role of the NH2-terminal region of the 402-residue-long beta-1,4-galactosyltransferase (beta-1,4-GT), a series of mutants and chimeric cDNA were constructed by polymerase chain reaction and transiently expressed in COS-7 cells, the enzyme activities were measured, and the protein was localized in the cells by subcellular fractionation or indirect immunofluorescence microscopy. We showed earlier that the deletion of the amino-terminal cytoplasmic tail and transmembrane domain from GT abolishes the stable expression of this protein in mammalian cells (Masibay, A.S., Boeggeman, E., and Qasba, P.K. (1992) Mol. Biol. Rep. 16, 99-104). Further deletion analyses of the amino-terminal region show that the first 21 amino acids of beta-1,4-GT are not essential for the stable production of the protein and are consistently localized in the Golgi apparatus. In addition, analysis of hybrid constructs showed that residues 1-25 of alpha-1,3-galactosyltransferase can functionally replace the beta-1,4-GT amino-terminal domain (residues 1-43). This fusion protein also showed Golgi localization. On the other hand, the alpha-2,6-sialyltransferase/beta-1,4-GT fusion protein (alpha-2,6-ST/beta-1,4-GT) needed additional COOH-terminal sequences flanking the transmembrane domain of the alpha-2,6-ST for stability and Golgi localization. Substitution of Arg-24, Leu-25, Leu-26, and His-33 of the beta-1,4-GT transmembrane by Ile (pLFM) or substitution of Tyr by Ile at positions 40 and 41 coupled with the insertion of 4 Ile residues at position 43 (pLB) released the mutant proteins from the Golgi and was detected on the cell surface. Our results show that (a) the transmembrane domains of beta-1,4-GT, alpha-1,3-galactosyltransferase, and alpha-2,6-ST, along with its stem region, all play a role in Golgi targeting and participate in a common mechanism that allows the protein to be processed properly and not be degraded in vivo; (b) increasing the length of the transmembrane domain overrides the Golgi retention signal and directs the enzyme to the plasma membrane; and (c) the length of the hydrophobic region of the transmembrane domain of beta-1,4-GT is an important parameter but is not sufficient by itself for Golgi retention.

Computer modeling studies on the binding of 2',5'-linked dinucleoside phosphates to ribonuclease T1-influence of subsite interactions on the substrate specificity.

The modes of binding of Gp(2',5')A, Gp(2',5')C, Gp(2',5')G and Gp(2',5')U to RNase T1 have been determined by computer modelling studies. All these dinucleoside phosphates assume extended conformations in the active site leading to better interactions with the enzyme. The 5'-terminal guanine of all these ligands is placed in the primary base binding site of the enzyme in an orientation similar to that of 2'-GMP in the RNase T1-2'-GMP complex. The 2'-terminal purines are placed close to the hydrophobic pocket formed by the residues Gly71, Ser72, Pro73 and Gly74 which occur in a loop region. However, the orientation of the 2'-terminal pyrimidines is different from that of 2'-terminal purines. This perhaps explains the higher binding affinity of the 2',5'-linked guanine dinucleoside phosphates with 2'-terminal purines than those with 2'-terminal pyrimidines. A comparison of the binding of the guanine dinucleoside phosphates with 2',5'- and 3',5'-linkages suggests significant differences in the ribose pucker and hydrogen bonding interactions between the catalytic residues and the bound nucleoside phosphate implying that 2',5'-linked dinucleoside phosphates may not be the ideal ligands to probe the role of the catalytic amino acid residues. A change in the amino acid sequence in the surface loop region formed by the residues Gly71 to Gly74 drastically affects the conformation of the base binding subsite, and this may account for the inactivity of the enzyme with altered sequence i.e., with Pro, Gly and Ser at positions 71 to 73 respectively. These results thus suggest that in addition to recognition and catalytic sites, interactions at the loop regions which constitute the subsite for base binding are also crucial in determining the substrate specificity.

Modes of binding of 2'-AMP to RNase T1. A computer modeling study.

The modes of binding of adenosine 2'-monophosphate (2'-AMP) to the enzyme ribonuclease (RNase) T1 were determined by computer modelling studies. The phosphate moiety of 2'-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites--(1) The primary base binding site where the guanine of 2'-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3'-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2'-AMP to RNase T1 were found to be of nearly the same energy implying that in solution 2'-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T1-2'-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T1 catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2'-AMP and 2'-GMP with RNase T1 reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme-2'-GMP complex.

Computer modeling studies on the subsite interactions of ribonuclease T1.

The modes of binding of pGp,ApG,CpG and UpG to the enzyme ribonuclease T1 were determined by computer modeling. Essentially two binding modes are possible for all the four ligands--one with the 3'-phosphate group occupying the phosphate binding site (substrate mode of binding) and the second with the 5'-phosphate group occupying the phosphate binding site (inhibitor mode of binding). The latter binding mode is energetically favoured over the former and in this mode the base (G) and the 5'-phosphate moieties occupy the same sites on the enzyme as 5'-GMP when bound to RNase T1. The ribose moiety of pGp adopts a C3'-endo pucker form when bound to the enzyme and the glycosyl torsion angle will be in -syn range as 5'-GMP in the RNase T1-5'-GMP complex. Based on these results, a mechanism for the release of the product subsequent to cleavage of the substrate by the enzyme has been proposed. The amino acid residues Asn98 and Tyr45 are shown to form the subsites for the phosphate and the base respectively on the 5'-side of the guanine occupying the primary binding site. These studies also provide a stereochemical explanation for the specificity of the 1N subsite for adenine.

Computer modelling studies of ribonuclease T1-2'-deoxy-2'-fluoroguanylyl- (3',5')-cytidine complex.

The mode of binding of the substrate analog 2'-deoxy-2'-fluoroguanylyl- (3',5')-cytidine (GfpC) to RNase T1 was determined by computer modelling studies. The results obtained are in good agreement with the observations of 1H-nmr studies. The modes of binding of the substrate analog GfpC and the substrate GpC to the enzyme RNase T1 have been compared. Though the guanine base favours to occupy the same site of the enzyme in both the complexes, significant differences are observed in the local environment around the 2'-substituent group of guanosine ribose moiety. In the RNase T1-GpC complex, the 2'-OH group is in close proximity to the side chain carboxylic acid of Glu58 which leads to the formation of a hydrogen bond. However, in the RNase T1-GfpC complex, 2'-fluorine is positioned away from Glu58 due to electrostatic repulsion and instead forms a hydrogen bond with His40 imidazolium group. The results obtained rule out the possibility of His40 serving as the base group in catalysis as suggested by 1H-nmr studies and further support the primary role assigned to Glu58 as the general base group by earlier computer modelling and the recent site directed mutagenesis studies. This study also implies that the 2'-deoxy-2'-fluoro substrate analog may not serve as a good model for determining the amino acid residue which serves as the general base group in ribonuclease catalysed reactions.

Computer modelling studies on the mechanism of action of ribonuclease T1.

The mechanism of action of ribonuclease (RNase) T1 is still a matter of considerable debate as the results of x-ray, 2-D nmr and site-directed mutagenesis studies disagree regarding the role of the catalytically important residues. Hence computer modelling studies were carried out by energy minimisation of the complexes of RNase T1 and some of its mutants (His40Ala, His40Lys, and Glu58Ala) with the substrate guanyl cytosine (GpC), and of native RNase T1 with the reaction intermediate guanosine 2',3'-cyclic phosphate (G greater than p). The puckering of the guanosine ribose moiety in the minimum energy conformer of the RNase T1-GpC (substrate) complex was found to be O4'-endo and not C3'-endo as in the RNase T1-3'-guanylic acid (inhibitor/product) complex. A possible scheme for the mechanism of action of RNase T1 has been proposed on the basis of the arrangement of the catalytically important amino acid residues His40, Glu58, Arg77, and His92 around the guanosine ribose and the phosphate moiety in the RNase T1-GpC and RNase T1-G greater than p complexes. In this scheme, Glu58 serves as the general base group and His92 as the general acid group in the transphosphorylation step. His40 may be essential for stabilising the negatively charged phosphate moiety in the enzyme-transition state complex.

Computer modeling studies of ribonuclease T1-guanosine monophosphate complexes.

The three-dimensional structures of ribonuclease (RNase) T1 complexes with the inhibitors 2'-guanylic acid (2'-GMP), 3'-guanylic acid (3'-GMP), and 5'-guanylic acid (5'-GMP) were predicted by energy minimization studies. It is shown that these inhibitors can bind to RNase T1 in either of the ribose puckered conformations (C2'-endo and C3'-endo) in solid state and exist in significant amounts in both forms in solution. These studies are in agreement with the x-ray crystallographic studies of the 2'-GMP-Lys25-RNase T1 complex, where the inhibitor binds in C2'-endo puckered conformation. These results are also in good agreement with the available 1H-nmr results of Inagaki et al. [(1985) Biochemistry 24, 1013-1020], but differ from their conclusions where the authors favor only the C3'-endo ribose conformation for all the three inhibitors. The calculations explain the apparent discrepancies in the conclusions drawn by x-ray crystallographic and spectroscopic studies. An extensive hydrogen-bonding scheme was predicted in all the three complexes. The hydrogen-bonding scheme predicted for the 2'-GMP (C2'-endo)-RNase T1 complex agrees well with those reported from x-ray crystallographic studies. In all three complexes the base and the phosphate bind in nearly identical sites independent of the position of the phosphate or the ribose pucker. The glycosyl torsion angle favors a value in the +syn range in the 2'-GMP (C2'-endo)-RNase T1, 3'-GMP (C2'-endo)-RNase T1, and 3'-GMP (C3'-endo)-RNase T1 complexes; in the high-syn range in the 2'-GMP (C3'-endo)-RNase T1 complex; and in the -syn range in the 5'-GMP (C2'-endo)-RNase T1 and 5'-GMP (C3'-endo)-RNase T1 complexes. These results are in agreement with experimental studies showing that the inhibitory power decreases in the order 2'-GMP greater than 3'-GMP greater than 5'-GMP, and they also explain the high pKa value observed for Glu58 in the 2'-GMP-RNase T1 complex.