Estimation of effective concentrations enforced by complex linker architectures from conformational ensembles

Abstract

Proteins and protein assemblies often tether interaction partners to strengthen interactions, to regulate activity through auto-inhibition or -activation, or to boost enzyme catalysis. Tethered reactions are regulated by the architecture of tether, which define an effective concentration of the interactors. Effective concentrations can be estimated theoretically for simple linkers via polymer models, but there is currently no general method for estimating effective concentrations for complex linker architectures consisting of both flexible and folded domains. We describe how effective concentrations can be estimated computationally for any protein linker architecture by defining a realistic conformational ensemble. We benchmark against prediction from a worm-like chain and values measured by competition experiments, and find minor differences likely due to excluded volume effects. Systematic variation of the properties of flexible and folded segments show that the effective concentration is mainly determined by the combination of the total length of flexible segments and the distance between termini of the folded domains. We show that a folded domain in a disordered linker can increase the effective concentration beyond what can be achieved by a fully disordered linker by focusing the end-to-end distance at the appropriate spacing. This suggest that complex linker architecture may have advantages over simple flexible linker, and emphasize that annotation as a linker should depend on the molecular context.

The optimal docking strength for reversibly tethered kinases

“…The effective concentration was determined from an ensemble of 10.000 conforms where MBD2:p66α is simulated as a rigid rod, and disordered segments are treated as a random chain (Fig S3). 25 For the 120-residue GS-linker this resulted in a calculated C eff of 388 μM. The numerical simulations predict that steady-state phosphorylation rates have a bell-shaped dependence on the docking strength ( k off ) for substrate concentrations from 0.1 to 100 μM (Fig.…”

“…To calculate C eff covering this complexity, a newly developed approach based on conformational ensembles was used. 25 Briefly, a physically realistic ensemble of the linker was modelled using the “Ensemble Optimization Method” (EOM). As EOM cannot handle heterodimers, the p66a-MBD2 coiled-coil was replaced with a α-helical poly-alanine rod, which has a similar length and produces the same end-to-end distribution as the folded domain.…”

“…As EOM cannot handle heterodimers, the p66a-MBD2 coiled-coil was replaced with a α-helical poly-alanine rod, which has a similar length and produces the same end-to-end distribution as the folded domain. 25 Flexible segments were simulated as beads using the “Native chain” for an ensemble of 10.000 conformations. The flexible segments were defined from the first residue not defined in the PDB structures of the PKAc (PDB: 2CPK) or the p66a:MBD2 coiled-coil (PDB: 2L2L) and the first residue in the consensus substrate motif.…”

Thumb-domain dynamics modulate the functional repertoire of DNA-Polymerase IV (DinB)

“…Besides orienting the PAD domain at the right distance to bind a double stranded DNA as shown by previous crystallographic studies 14,60,61 , another intriguing possibility is the effective concentration of this domain. It could be shown previously for unrelated enzymes, kinases that facilitate phosphorylation of their substrates, that in cases when the enzyme is tethered to the substrate these linker regions have a direct influence on the catalytic rate 62,63 . In agreement with this general interpretation of the importance of the linker region already previous work on chimeric archaeal DinB homologous protein with varying linker sequences high-lighted the importance of this linker region for the catalytic efficiency of the enzyme 15,26 .…”

Recently Designed Multivalent Spike Binders Cannot Bind Multivalently─How Do They Achieve Enhanced Avidity to SARS-CoV-2?

“…Kjaergaard 24 developed an ensemble approach to model the effect of complex linker architectures on bivalent avidity. This analysis confirmed the findings of Galimidi et al 22 that a rigid linker, tuned to the distance and flanked by short flexible segments, could give enhanced avidity relative to a fully flexible linker.…”