Research Assistant University of Rhode Island Wakefield, Rhode Island
Body of Abstract: Cellulose synthases (CESAs) are transmembrane proteins that polymerize glucose on their cytoplasmic side and transport the resulting glucan chains to the extracellular matrix through integral transmembrane (TM) channels. To investigate interactions between nascent glucan chains and amino acids lining the TM channels, we predicted interactions by computational modeling and tested them using an assay based on complementation of the Physcomitrium patens cesa5 knockout (KO) mutation, which results in an easily scored ‘no gametophore’ phenotype. Using an all-atom molecular dynamics simulation of a PpCESA5 trimer, contact analysis was performed to identify amino acids that interact with glucan chains as they pass through the TM channels of a homo-oligomeric cellulose synthesis complex (CSC). Point mutations predicted to interfere with interactions between glucan chains and amino acids lining the TM channel, without disrupting TM helix structure, were made in PpCESA5 through site directed mutagenesis. Vectors containing mutated ppcesa5 genes were tested for complementation by constitutive expression in background lines that permit their integration into homo-oligomeric (ppcesa5/6/7KO) or hetero-oligomeric (ppcesa5KO) CSCs. Loss of single predicted glucan-interacting amino acid side chains abolished or impaired PpCESA5 function in ppcesa5/6/7KO, consistent with disruption of a smooth energy profile within the TM channel. Mutations eliminating polar or charged amino acid side chains had the most severe effects. Mutations eliminating aromatic side chains had milder effects and altering non-polar, non-aromatic side chains had little or no effect on PpCESA5 function. Vectors that failed to rescue the phenotype in ppcesA5/6/7KO were transformed into ppcesA5 KO, which permits integration of the mutated CESAs into hetero-oligomeric CSCs with wild type subunits. This resulted in cell expansion phenotypes consistent with disruption of normal microfibril formation. We are investigating whether these mutations alter microfibril structure and digestibility.