Lars C. Pedersen

Crystal structure of estrogen sulphotransferase.

The structure of estrogen sulphotransferase has been solved in the presence of inactive cofactor PAP and substrate 17β-estradiol. This structure reveals structural similarities between cytosolic sulphotransf erases and nucleotide kinases.

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I.

Human β1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Galβ1–3Galβ1–4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 Å in the presence of the donor substrate product UDP, the catalytic Mn2+ ion, and the acceptor substrate analog Galβ1–3Galβ1–4Xyl. The enzyme is a α/β protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu227, Asp252, and Glu281 dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu281 is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp194, Asp195, Asp196) has direct interaction with the ribose of the UDP molecule as well as with the Mn2+ ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.

A synergistic approach to protein crystallization: Combination of a fixed-arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

Crystal Structure of the Sulfotransferase Domain of Human Heparan Sulfate N-Deacetylase/ N-Sulfotransferase 1

Heparan sulfateN-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-Å resolution in a binary complex with 3′-phosphoadenosine 5′-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single α/β fold with a central five-stranded parallel β-sheet and a three-stranded anti-parallel β-sheet bearing an interstrand disulfide bond. The structural regions α1, α6, β1, β7, 5′-phosphosulfate binding loop (between β1 and α1), and a random coil (between β8 and α13) constitute the PAP binding site of NST1. The α6 and random coil (between β2 and α2), which form an open cleft near the 5′-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5′-phosphate.

A structural solution for the DNA polymerase λ-dependent repair of DNA gaps with minimal homology

Human DNA polymerase lambda (Pol λ) is a family X member with low frameshift fidelity that has been suggested to perform gap-filling DNA synthesis during base excision repair and during repair of broken ends with limited homology. Here, we present a 2.1 A crystal structure of the catalytic core of Pol λ in complex with DNA containing a two nucleotide gap. Pol λ makes limited contacts with the template strand at the polymerase active site, and superimposition with Pol β in a ternary complex suggests a shift in the position of the DNA at the active site that is reminiscent of a deletion intermediate. Surprisingly, Pol λ can adopt a closed conformation, even in the absence of dNTP binding. These observations have implications for the catalytic mechanism and putative DNA repair functions of Pol λ.

The Sulfuryl Transfer Mechanism CRYSTAL STRUCTURE OF A VANADATE COMPLEX OF ESTROGEN SULFOTRANSFERASE AND MUTATIONAL ANALYSIS

Estrogen sulfotransferase (EST) catalyzes transfer of the 5′-sulfuryl group of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) to the 3α-phenol group of estrogenic steroids such as estradiol (E2). The recent crystal structure of EST-adenosine 3′,5′-diphosphate (PAP)- E2complex has revealed that residues Lys48, Thr45, Thr51, Thr52, Lys106, His108, and Try240 are in position to play a catalytic role in the sulfuryl transfer reaction of EST (Kakuta Y., Pedersen, L. G., Carter, C. W., Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904–908). Mutation of Lys48, Lys106, or His108 nearly abolishes EST activity, indicating that they play a critical role in catalysis. A present 2.2-Å resolution structure of EST-PAP-vanadate complex indicates that the vanadate molecule adopts a trigonal bipyramidal geometry with its equatorial oxygens coordinated to these three residues. The apical positions of the vanadate molecule are occupied by a terminal oxygen of the 5′-phosphate of PAP (2.1 Å) and a possible water molecule (2.3 Å). This water molecule superimposes well to the 3α-phenol group of E2 in the crystal structure of the EST·PAP·E2 complex. These structures are characteristic of the transition state for an in-line sulfuryl transfer reaction from PAPS to E2. Moreover, residues Lys48, Lys106, and His108 are found to be coordinated with the vanadate molecule at the transition state of EST.

Replication infidelity via a mismatch with Watson–Crick geometry

In describing the DNA double helix, Watson and Crick suggested that “spontaneous mutation may be due to a base occasionally occurring in one of its less likely tautomeric forms.” Indeed, among many mispairing possibilities, either tautomerization or ionization of bases might allow a DNA polymerase to insert a mismatch with correct Watson–Crick geometry. However, despite substantial progress in understanding the structural basis of error prevention during polymerization, no DNA polymerase has yet been shown to form a natural base–base mismatch with Watson–Crick-like geometry. Here we provide such evidence, in the form of a crystal structure of a human DNA polymerase λ variant poised to misinsert dGTP opposite a template T. All atoms needed for catalysis are present at the active site and in positions that overlay with those for a correct base pair. The mismatch has Watson–Crick geometry consistent with a tautomeric or ionized base pair, with the pH dependence of misinsertion consistent with the latter. The results support the original idea that a base substitution can originate from a mismatch having Watson–Crick geometry, and they suggest a common catalytic mechanism for inserting a correct and an incorrect nucleotide. A second structure indicates that after misinsertion, the now primer-terminal G•T mismatch is also poised for catalysis but in the wobble conformation seen in other studies, indicating the dynamic nature of the pathway required to create a mismatch in fully duplex DNA.

Crystal structure of SULT2A3, human hydroxysteroid sulfotransferase.

The crystal structure of SULT2A3 human hydroxysteroid sulfotransferase has been solved at 2.4 Å resolution in the presence of 3′‐phosphoadenosine 5′‐phosphate (PAP). The overall structure is similar to those of SULT1 enzymes such as estrogen sulfotransferase and the PAP binding site is conserved, however, significant differences exist in the positions of loops Pro14–Ser20, Glu79–Ile82 and Tyr234–Gln244 in the substrate binding pocket. Moreover, protein interaction in the crystal structure has revealed a possible dimer‐directed conformational alteration that may regulate the SULT activity.

Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana

The receptor for activated C‐kinase 1 (RACK1) is a highly conserved WD40 repeat scaffold protein found in a wide range of eukaryotic species from Chlamydymonas to plants and humans. In tissues of higher mammals, RACK1 is ubiquitously expressed and has been implicated in diverse signaling pathways involving neuropathology, cellular stress, protein translation, and developmental processes. RACK1 has established itself as a scaffold protein through physical interaction with a myriad of signaling proteins ranging from kinases, phosphatases, ion channels, membrane receptors, G proteins, IP3 receptor, and with widely conserved structural proteins associated with the ribosome. In the plant Arabidopsis thaliana, RACK1A is implicated in diverse developmental and environmental stress pathways. Despite the functional conservation of RACK1‐mediated protein–protein interaction‐regulated signaling modes, the structural basis of such interactions is largely unknown. Here we present the first crystal structure of a RACK1 protein, RACK1 isoform A from Arabidobsis thaliana, at 2.4 Å resolution, as a C‐terminal fusion of the maltose binding protein. The structure implicates highly conserved surface residues that could play critical roles in protein–protein interactions and reveals the surface location of proposed post‐transcriptionally modified residues. The availability of this structure provides a structural basis for dissecting RACK1‐mediated cellular signaling mechanisms in both plants and animals.

Enzymatic redesigning of biologically active heparan sulfate

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3′-phosphoadenosine 5′-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.