Structural basis for heparan sulfate co-polymerase action by the EXT1-2 complex

Area of Science:

• Biochemistry
• Glycobiology
• Structural Biology

Background:

• Heparan sulfate (HS) proteoglycans are crucial extracellular matrix components involved in cell signaling and development.
• HS biosynthesis is mediated by the obligate EXT1-EXT2 heterocomplex, with each protein possessing distinct glycosyltransferase domains.
• Understanding the structural basis of HS synthesis is vital for deciphering its biological roles and therapeutic potential.

Purpose of the Study:

• To determine the structure of the human EXT1-EXT2 heterocomplex bound to substrates.
• To elucidate the specific glycosyltransferase domains responsible for HS co-polymer synthesis.
• To investigate the mechanism of HS polymerization by the EXT1-EXT2 complex.

Main Methods:

• Generation of a functional human EXT1-EXT2 heterocomplex.
• Determination of the complex's structure using X-ray crystallography.
• Enzymatic activity assays using catalytic site mutants of EXT1 and EXT2.

Main Results:

• The structure of the human EXT1-EXT2 heterocomplex was determined with bound donor and acceptor substrates.
• Only two glycosyltransferase domains, EXT1's GT-B fold (β1,4GlcA transferase) and EXT2's GT-A fold (α1,4GlcNAc transferase), are essential for HS co-polymer synthesis.
• These two active sites are located over 90 Å apart, suggesting a dissociative synthesis mechanism.

Conclusions:

• Heparan sulfate polymerization is primarily driven by two specific glycosyltransferase domains within the EXT1-EXT2 complex.
• The synthesis of heparan sulfate likely proceeds through a dissociative mechanism involving separate catalytic events.
• This structural and mechanistic insight provides a foundation for understanding HS-related biological processes and developing targeted therapies.