Tied to Work: Topologically Knotted SPOUT Methyltransferases and UCH Deubiquitinases in Cellular Biochemistry

Topologically knotted proteins are emerging as functional elements of the proteome rather than structural curiosities. Among the recurring protein knot types, the 3₁ trefoil, 5₂ Gordian and 7₁ septoil knots provide useful models for understanding how topology may reshape catalysis, substrate recognition, folding and mechanical behaviour. This review focuses on two systems in which these links can already be made with unusual clarity: the 3₁-knotted SPOUT methyltransferase superfamily and the 5₂-knotted ubiquitin C-terminal hydrolases (UCHs). In SPOUT methyltransferases, the knot defines a compact S-adenosyl-l-methionine (SAM)-binding pocket and enforces the bent cofactor geometry required for efficient methyl transfer. In UCH deubiquitinases, the 5₂ knot stabilises catalytic architecture, constrains the crossover loop that gates substrate access, and helps explain how mutations, regulatory elements and disease-linked perturbations alter function. Beyond catalysis, knotted topology may also modulate how some proteins respond to force, although the range of mechanostabilities across the knotted proteome is broad and many knotted proteins show moderate resistance to mechanical unfolding. Comparisons between 5₂-knotted UCH paralogs and the 7₁-knotted protein Q9PR55 raise the possibility that knot geometry contributes to resilience to withstand vectorial pulling, that is, mechanical unfolding, by a AAA+ protease, ClpXP, alongside local folding stability. In the context of folding mechanisms, knotted proteins are challenging systems in that many populate long-lived intermediates or misthreaded states along parallel or bifurcated folding pathways. Although populated intermediates and slow folding are also well documented in numerous unknotted systems, the question of how knotted topologies contribute to the formation of folding intermediates together with knotting/threading events adds to an extra layer of complexity to the analyses. Together, these examples support a broader view in which topology is not merely descriptive geometry, but a mechanistic variable that links structure, dynamics and biological function.