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Biomedical subjects

V Sasisekharan

Publications and source records attributed to V Sasisekharan.

At least 19 recordsLinked to original sources
PubMed2005

Structural insights into biological roles of protein-glycosaminoglycan interactions.

The extracellular environment is largely comprised of complex polysaccharides, which were historically considered inert materials that hydrated the cells and contributed to the structural scaffolds. Recent advances in development of sophisticated analytical techniques have brought about a dramatic transformation in understanding the numerous biological roles of these complex polysaccharides. Glycosaminoglycans (GAGs) are a class of these polysaccharides, which bind to a wide variety of proteins and signaling molecules in the cellular environment and modulate their activity, thus impinging on fundamental biological processes. Despite the importance of GAGs modulating biological functions, there are relatively few examples that demonstrate specificity of GAG-protein interactions, which in turn define the structure-function relationships of these polysaccharides. Focusing on heparin/heparan (HSGAGs) and chondroitin/dermatan sulfate (CSGAGs), this review provides structural insights into the oligosaccharide-protein interactions and discusses some key and challenging aspects of understanding GAG structure-function relationships.

Animals↗
PubMed2003

Structural specificity of heparin binding in the fibroblast growth factor family of proteins.

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) mediate a wide variety of complex biological processes by specifically binding proteins and modulating their biological activity. One of the best studied model systems for protein-HSGAG interactions is the fibroblast growth factor (FGF) family of molecules, and recent observations have demonstrated that the specificity of a given FGF ligand binding to its cognate receptor (FGFR) is mediated by distinct tissue-specific HSGAG sequences. Although it has been known that sulfate and carboxylate groups in the HSGAG chain play a key role by interacting with basic residues on the proteins, there is little understanding of how these ionic interactions provide the necessary specificity for protein binding. In this study, using all of the available crystal structures of different FGFs and FGF-HSGAG complexes, we show that in addition to the ionic interactions, optimal van der Waals contact between the HSGAG oligosaccharide and the protein is also very important in influencing the specificity of FGF-HSGAG interactions. Although the overall helical structure is maintained in the FGF-bound HSGAG compared with unbound HSGAG, we observe distinct changes in the backbone torsion angles of the oligosaccharide chain induced upon protein binding. These changes result in local deviations in the helical axis that provide optimal ionic and van der Waals contact with the protein. A specific conformation and topological arrangement of the HSGAG-binding loops of FGF, on the other hand, impose structural constraints that induce the local deviations in the HSGAG structure, thereby enabling maximum contact between HSGAG and the protein.

Binding Sites↗
PubMed2002

Identification of structural motifs and amino acids within the structure of human heparan sulfate 3-O-sulfotransferase that mediate enzymatic function.

In an accompanying paper [J. R. Myette, Z. Shriver, J. Liu, G. Venkataraman, and R. Sasisekharan (2002) Biochem. Biophys. Res. Commun. 290, 1206-1213], we described the purification and biochemical characterization of a soluble, recombinantly expressed form of the human heparan sulfate 3-O-sulfotransferase (3-OST-1). Such an important first step enables detailed structure-function studies for this class of enzymes. Herein, we describe a complimentary, structure-based homology modeling approach for predicting 3-OST-1 structure. This approach employs a variety of structural analysis and molecular modeling tools used in conjunction with protein crystallographic studies of related enzymes. In this manner, we describe important motifs within the predicted three-dimensional structure of the enzyme and identify specific amino acids that are likely important for enzymatic function.

Adenosine Diphosphate↗
PubMed1999

Molecular characteristics of fibroblast growth factor-fibroblast growth factor receptor-heparin-like glycosaminoglycan complex.

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF-FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF-FGFR interaction mediated by the 'conserved' primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the 'variable' secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1beta receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF-FGFR interactions. In the FGF-FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

Amino Acid Sequence↗
PubMed1997

Mixed backbone antisense oligonucleotides: design, biochemical and biological properties of oligonucleotides containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments.

We have designed and synthesized mixed backbone oligonucleotides (MBOs) containing 2'-5'-ribo- and 3'-5'-deoxyribonucleotide segments. Thermal melting studies of the phosphodiester MBOs (three 2'-5'linkages at each end) with the complementary 3'-5'-DNA and -RNA target strands suggest that 2'-5'-ribonucleoside incorporation into 3'-5'-oligodeoxyribonucleotides reduces binding to the target strands compared with an all 3'-5'-oligodeoxyribonucleotide of the same sequence and length. Increasing the number of 2'-5'linkages (from six to nine) further reduces binding to the DNA target strand more than the RNA target strand [Kandimalla,E.R. and Agrawal,S. (1996)Nucleic Acids Symp. Ser., 35, 125-126]. Phosphorothioate (PS) analogs of MBOs destabilize the duplex with the DNA target strand more than the duplex with the RNA target strand. Circular dichroism studies indicate that the duplexes of MBOs with the DNA and RNA target strands have spectral characteristics of both A- and B-type conformations. Compared with the control oligonucleotide, MBOs exhibit moderately higher stability against snake venom phosphodiesterase, S1 nuclease and in fetal calf serum. Although 2'-5'modification does not evoke RNase H activity, this modification does not effect the RNase H activation property of the 3'-5'-deoxyribonucleotide segment adjacent to the modification. In vitro studies with MBOs suggest that they have lesser effects on cell proliferation, clotting prolongation and hemolytic complement lysis than do control PS oligodeoxyribonucleotides. PS analogs of MBOs show HIV-1 inhibition comparable with that of a control PS oligodeoxyribonucleotide with all 3'-5'linkages. The current results suggest that a limited number of 2'-5'linkages could be used in conjunction with PS oligonucleotides to further modulate the properties of antisense oligonucleotides as therapeutic agents.

Animals↗
PubMed1997

Single-stranded DNA and RNA targeted triplex-formation: UV, CD and molecular modeling studies of foldback triplexes containing different RNA, 2'-OMe-RNA and DNA strand combinations.

We studied the influence of different 2'-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D.D:D, R*.D:D, D.D:R*, R*.D:R*, D.R:D, R*.R:D, D.R:R*, and R*.R:R*; where the first, middle, and last letters stand for the Hoogsteen Pyrimidine, Watson-Crick [WC] purine and WC pyrimidine strands, respectively, and D, R and R* stand for DNA, RNA and 2'-OMe-RNA strands, respectively) indicate more stable foldback triplex formation with a DNA purine strand than with an RNA purine strand. Of the four possible WC duplexes with RNA/DNA combinations, the duplex with a DNA purine strand and a 2'-O-Me-RNA pyrimidine strand forms the most thermally stable triplex, although its thermal stability is the lowest of all four duplexes. Irrespective of the duplex combination, a 2'-OMe-RNA Hoogsteen pyrimidine strand forms a stable foldback triplex over a DNA Hoogsteen pyrimidine strand confirming the earlier reports with conventional and circular triplexes. The CD studies suggest a B-type conformation for an all DNA homo-foldback triplex (D.D:D), while hetero-foldback triplex spectra suggest intermediate conformation to both A-type and B-type structures. A novel molecular modeling study has been carried out to understand the stereochemical feasibility of all the combinations of foldback triplexes using a geometric approach. The new approach allows use of different combinations of chain geometries depending on the nature of the chain (RNA vs. DNA).

Circular Dichroism↗
PubMed1996

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Apoproteins↗
PubMed1996

Structure of Py.Pu.Py DNA triple helices. Fourier transforms of fiber-type x-ray diffraction of single crystals.

Well-formed hexagonal crystals of oligomeric DNA triple helices exhibit fiber-type x-ray diffraction patterns [cf., Liu et al. (1994) Nature Struct. Biol. 1, 11], which can be interpreted in terms of Fourier transforms of these helices. Precession photographs of a triplex formed of dA and dT chains show that it has 13 residues per turn. In contrast, a sequence containing the four natural bases A, G, C, and T has 12 residues per turn. In this sense the triple helices exhibit a sequence-dependent polymorphism, though both have C2'-endo sugar pucker and B rather than A conformation. New models are constructed, using constraints from x-ray diffraction, and Fourier transforms of the models are calculated. Good agreement in the amplitudes and positions of the calculated and observed diffraction intensities confirms the structures for both triple helices. These are the first stereochemically satisfactory DNA triple helices for which coordinates based on adequate experimental data were provided. Sequences for crystallization are designed to achieve unique base alignments and are screened for the presence of sharp bands on gel electrophoresis to assure the absence of multiple species caused by strand slippage. Despite intensive efforts to observe normal crystal diffraction by varying sequences and conditions, all crystals exhibited only fiber-type diffraction. We suggest that this behavior may be an intrinsic property of triple helices and discuss possible reasons for the results. Spectroscopic and chemical experiments establish that the oligonucleotides exist in solution as triple helices under the conditions of crystallization.

Computer Simulation↗
PubMed1995

Single strand targeted triplex formation: targeting purine-pyrimidine mixed sequences using abasic linkers.

Foldback triplex-forming oligonucleotides (FTFOs) that contain an abasic linker, [2-(4-aminobutyr-1-yl)-1,3-propanediol] (APD linker), in the Hoogsteen domain against pyrimidine bases of a C:G and a T:A base pair were studied for their relative stability and sequence specificity of triplex formation. In general, the APD linker has less destabilizing effect against a C:G base pair than a T:A base pair. Incorporation of three APD linker moieties resulted in decreased binding to the target, which was comparable to results observed with three imperfectly matched natural base triplets. The APD linker incorporation did not result in the loss of sequence specificity of FTFOs, unlike in the case of normal triplex-forming oligonucleotides (TFOs). The introduction of a positively charged abasic linker, however, resulted in decreased stability of the triplex, because of loss of hydrogen bonding and stacking interactions in the major groove. The results of a molecular modeling study show that APD linker can be readily incorporated without any change in the conformation of the natural sugar-phosphate backbone conserving overall triple helix geometry. Further, the modeling study suggests a hydrogen bond formation between the amino group of linker and N4 of cytosine mediated by a solvent molecule (water) in the floor of the base triplet in addition to a contribution from the positive charge on the APD linker amino group. Either a direct or water-mediated hydrogen bond between the amino group of the APD linker and the O4 of thymine is unlikely when the linker is placed against a T:A base pair.

Base Composition↗
PubMed1995

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Amino Acid Sequence↗
PubMed1995

Symmetry and structure of RNA and DNA triple helices.

Despite wide interest in nucleic acid triple helices, there has been no stereochemically satisfactory structure of an RNA triple helix in atomic detail. AN RNA triplex structure has previously been proposed based on fiber diffraction and molecular modeling [S. Arnott and P. J. Bond (1973) Nature New Biology, Vol. 244, pp. 99-101; S. Arnott, P. J. Bond, E. Selsing, and P. J. C. Smith (1976) Nucleic Acids Research, Vol. 3, pp. 2459-2470], but it has nonallowed close contacts at every triplet and is therefore not stereochemically acceptable. We propose here a new model for an RNA triple helix in which the three chains have identical backbone conformations and are symmetry related. There are no short contacts. The modeling employs a novel geometrical approach using the linked atom least squares [P. J. C. Smith and S. Arnott (1978) Acta Crystallographica, Vol. A34, pp. 3-11] program and is not based on energy minimization. In general, the method leads to a range of possible structures rather than a unique structure. In the present case, however, the constraints resulting from the introduction of a third strand limit the possible structures to a very small range of conformation space. This method was used previously to obtain a model for DNA triple helices [G. Raghunathan, H. T. Miles, and V. Sasisekharan (1993) Biochemistry, Vol. 32, pp. 455-462], subsequently confirmed by fiber-type x-ray diffraction of oligomeric crystals [K. Liu, H. T. Miles, K. D. Parris, and V. Sasisekharan (1994) Nature Structural Biology, Vol. 1, pp. 11-12]. The above triple helices have Watson-Crick-Hoogsteen [K. Hoogsteen (1963) Acta Crystallographica, Vol. 16, pp. 907-916] pairing of the three bases. The same modeling method was used to investigate the feasibility of three-dimensional structures based on the three possible alternative hydrogen-bonding schemes: Watson-Crick-reverse Hoogsteen, Donohue [J. Donohue (1953) Proceedings of the National Academy of Science USA, Vol. 39, pp. 470-475] (reverse Watson-Crick)-Hoogsteen, and Donohue-reverse Hoogsteen. We found that none of these can occur in either RNA or DNA helices because they give rise only to structures with prohibitively short contacts between backbone and base atoms in the same chain.

DNA↗
PubMed1994

A stereochemical approach to pyranose ring flexibility: its implications for the conformation of dermatan sulfate.

Glycosaminoglycans, such as heparin, heparan sulfate, and dermatan sulfate, are characterized by a disaccharide repeating unit of a uronate and a hexosamine and are increasingly understood to be important physiologically as soluble components of the extracellular matrix. The secondary structure of this class of acidic polysaccharides is believed to play a key role in determining the wide range of biological specificities. Central to the structural diversity of the glycosaminoglycans is the experimentally documented conformational flexibility of the iduronate residue. Here, we outline an approach to explore the iduronate conformational flexibility by imposing stereochemical criteria of nonbonded contact distances. By performing a complete search of all possible torsions that define the iduronate ring geometry, we eliminate any prior bias with regard to minimum energy conformers. The approach led to alternative feasible conformers for the iduronate ring that are stereochemically satisfactory and are consistent with the available physico-chemical data.

Carbohydrate Conformation↗
PubMed1994

Parallel nucleic acid helices with Hoogsteen base pairing: symmetry and structure.

Molecular structures for parallel DNA and RNA double helices with Hoogsteen pairing are proposed for the first time. The DNA helices have sugars in the C2'-endo region and the phosphodiester conformations are (trans, gauche-), and the RNA helices have sugars in the C3'-endo region and the phosphodiester conformations are (gauche-, gauche-). A pseudorotational symmetry relates the two parallel strands of DNA helices and a screw symmetry relates the two strands of RNA helices, which have an associated tilt of the bases. The conformational space of parallel helices with Hoogsteen base pairing, unlike the Watson-Crick duplex, is highly restricted due to the unique positioning of the symmetry axis in the former case. The features of the parallel double helix with Hoogsteen pairing are compared with the Watson-Crick duplex and the corresponding triple helix.

Base Composition↗
PubMed1993

A novel DNA duplex. A parallel-stranded DNA helix with Hoogsteen base pairing.

We show here for the first time that a stable parallel double helix with Hoogsteen pairing can exist independently of the triple helix of which it is a component part. The experiments employ DNA oligonucleotides with mixed sequences of normal bases. These duplexes are distinct from previously reported ribopolynucleotide helices containing bulky substituents which prevent Watson-Crick pairing as well as from parallel duplexes with Donohue, or reversed Watson-Crick, pairing. Stoichiometry is established by mixing curves and gel electrophoresis. Tm depends linearly upon pH, increasing with acidity because of the need to protonate N3 of C. The Tm of the 20-mer studied here is 52 degrees C at pH 5.2 and 0.1 M NaCl. At pH above 6, the molecule rearranges to form an antiparallel duplex with imperfect Watson-Crick pairing and loops, and the Tm is then independent of pH. The CD spectrum of the parallel duplex is very similar to that of the corresponding triple helix but quite different from that of the Watson-Crick helix. The infrared spectrum in the double bond region closely resembles that of the triple helix but, as with the CD, is quite different from that of the Watson-Crick duplex. The infrared spectra of the duplex and triple helix are also nearly identical in the region form 800 to 1000 cm-1, which is sensitive to backbone conformation. The only symmetry element present is a pseudorotational axis coincident with the helix axis of the parallel duplex as well as with the axis of the corresponding triple helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1993

Symmetry and molecular structure of a DNA triple helix: d(T)n.d(A)n.d(T)n.

A structure for the triple helix d(T)n.d(A)n.d(T)n consistent with recent infrared spectral data is proposed, and its salient features are discussed. The present structure preserves the pseudodyad between the Watson-Crick base-paired adenine and thymine strands and in addition has a pseudorotational symmetry relating the Hoogsteen-paired adenine and thymine strands. The simultaneous presence of these two symmetries gives rise to a dyad between the two thymine polynucleotides. These symmetries result in identical backbone conformations for all three strands, unlike any previously proposed model for a triple helix. The proposed structure has an axial rise per residue of 3.26 A and 12 residues per turn obtained from X-ray fiber diffraction [Arnott S., & Selsing, E. (1974) J. Mol. Biol. 88, 509-521]. The present structure is structurally and conformationally similar to double helical B-form DNA and has sugar pucker in the C2'-endo region. This structure is fundamentally different from the one proposed by Arnott and co-workers, which was based on structural and conformational features similar to double helical A-form DNA with C3'-endo sugar pucker. It is stereochemically satisfactory, and it does not have the disallowed nonbonded distances present in the earlier model of Arnott and co-workers. It is energetically much more favorable than their structure. Coordinates of the present structure are given.

DNA↗
PubMed1992

Structure of d(T)n.d(A)n.d(T)n: the DNA triple helix has B-form geometry with C2'-endo sugar pucker.

The polynucleotide helix d(T)n.d(A)n.d(T)n is the only deoxypolynucleotide triple helix for which a structure has been published, and it is generally assumed as the structural basis for studies of DNA triplexes. The helix has been assigned to an A-form conformation with C3'-endo sugar pucker by Arnott and Selsing [1974; cf. Arnott et al. (1976)]. We show here by infrared spectroscopy in D2O solution that the helix is instead B-form and that the sugar pucker is in the C2'-endo region. Distamycin A, which binds only to B-form and not to A-form helices, binds to the triple helix without displacement of the third strand, as demonstrated by CD spectroscopy and gel electrophoresis. Molecular modeling shows that a stereochemically satisfactory structure can be build using C2'-endo sugars and a displacement of the Watson-Crick base-pair center from the helix axis of 2.5 A. Helical constraints of rise per residue (h = 3.26 A) and residues per turn (n = 12) were taken from fiber diffraction experiments of Arnott and Selsing (1974). The conformational torsion angles are in the standard B-form range, and there are no short contacts. In contrast, we were unable to construct a stereochemically allowed model with A-form geometry and C3'-endo sugars. Arnott et al. (1976) observed that their model had short contacts (e.g., 2.3 A between the phosphate-dependent oxygen on the A strand and O2 in the Hoogsteen-paired thymine strand) which are generally known to be outside the allowed range.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1992

Hairpin and parallel quartet structures for telomeric sequences.

The role of thymine residues in the formation of G-quartet structures for telomeric sequences has been investigated using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T3G4) and d(G4T4G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative bands while d(G4TG4) adopts a parallel G-quartet structure like d(G12) which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of hairpin G-quartet structures decreases with decrease in the number of intervening thymine residues. Potassium permanganate, a single strand specific probe has been used to establish the presence of loops composed of T residues in the hairpin G quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. The formation of hairpin G quartet structure for the above sequences is further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.

Animals↗