Insights into the N-sulfation mechanism: molecular dynamics simulations of the N-sulfotransferase domain of NDST1 and mutants.
Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS), yielding sulfated sugar chains and 3'-phosphoadenosine-5'-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.
Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, Brazil. tarsis.ferreira@cchmc.org
Tarsis F
TF
Gesteira
Laércio
L
Pol-Fachin
Vivien Jane
VJ
Coulson-Thomas
Marcelo A
MA
Lima
Hugo
H
Verli
Helena B
HB
Nader
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004187:Disaccharides / Q000737:chemistry; D006497:Heparan Sulfate / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry; D011485:Protein Binding; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics
D004187:Disaccharides; D006497:Heparan Sulfate; D050505:Mutant Proteins; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; C021350:heparitin sulfotransferase