Abstract:
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the primary step of photosynthetic carbon fixation but suffers from poor CO$_2$/O$_2$ substrate discrimination, limiting photosynthetic efficiency in C$_3$ crops by up to 25\%. Engineering improved specificity has remained a longstanding challenge, partly because the active site is structurally constrained and partly because specificity and catalytic turnover are inversely correlated across natural variants. An alternative strategy involves \emph{de novo} designed protein binders that allosterically modulate Rubisco large subunit (LSU) function as small subunit replacements. One such designed binder (binder77) confers greater specificity enhancement on the ancestral Rubisco variant e170N than the natural ancestral small subunit (ancSSU), yet the molecular basis for this superior effect remains unknown.Through this work, we aim to use molecular dynamics simulations paired with allosteric network analyses, followed by mutagenesis studies and their biochemical characterisation to identify potential interactions responsible for binder77's enhanced activity. The starting point for the molecular dynamics simulations were AlphaFold3-predicted structures, which have RMSD values of 0.32--0.35~ \AA against known crystal structures of Rubisco variants. The Shortest Path Map Tool used for allosteric analyses, revealed three reproducible interactions: a direct T316-L321-RuBP substrate coordination, a R125-D351 salt bridge, and a disruption of a L320-E510 interaction only seen in lower specificity variants. Three conservative point mutations: T316S, L321V, and D351N, were designed to test these interactions experimentally. All mutants expressed as correctly assembled, catalytically active L8 octamers and retained binder77 binding, confirming the structural integrity of the engineered variants. Together, these results establish a mechanistic framework for binder77 function and provide a foundation for rational second-generation binder design.
