Association pathways for and I5.6 (left) and I3.6 (right) are represented by the net flux from the unbound to the bound state.

Pathways are plotted along the forward committer probability, representing the probability to move forward from a given state. A total of 50 antibody VH/VL domain structures are shown for each state, representing the state encounter-ensemble. Structures were sampled from the MSMs, weighted by their stationary state probabilities. Arrow colors indicate the committer probability for the corresponding state, based on the VH/VL color of that state. Arrows indicate state connectivity for transitions toward the bound state with arrow weight representing the relative flux along transition paths. Numbers above each arrow indicate the percent contribution to the total transition flux for each transition.

(A) (left) Representative State 2 structure identified in the I3.6 and I5.6 MSMs. (right) Contact counts between I3.6 and I5.6 VH/VL antibody cleft and cleft proximal residues (Abc) with N332-glycan (N332g) D-arm residues and between antibody HCDR3 (AbH3) residues and residues near the N332-glycan base (N332b). (B) (left) Representative State 3 structure identified in the I3.6 and I5.6 MSMs. (right) Contact counts between I3.6 and I5.6 VH/VL antibody cleft and cleft proximal residues (Abc) with N332-glycan (N332g) D-arm residues and between antibody HCDR3 (AbH3) residues and residues near the N332-glycan base (N332b). (C) (left) Representative State 4 structure identified in the I3.6 and I5.6 MSMs. (right) Contact counts between I3.6 and I5.6 VH/VL antibody cleft and cleft proximal residues (Abc) with N332-glycan (N332g) D-arm residues and between antibody HCDR3 (AbH3) residues and residues near the N332-glycan base (N332b). Contacts represent the state MSM weighted mean number of contacts, defined as residues within 4.0 Å. Error bars represent the state MSM weighted contact standard deviation.

(A) Mean association rate constant value (n=3) heatmap for pairwise interactions between the I3.6 intermediate, I3.6 alanine mutants, and the mature DH270.6 antibody Fabs and CH848.d949 SOSIP and CH848.d949 alanine mutant SOSIPs. (B) Log mean dissociation rate constant value (n=3) heatmap for pairwise interactions between the I3.6 intermediate, I3.6 alanine mutants, and the mature DH270.6 antibody Fabs and CH848.d949 SOSIP and CH848.d949 alanine mutant SOSIPs. Asterisks indicate dissociation rate constants below the calibrated instrument detection threshold (<7e-6 s-1). Colors for these rate constants were assigned based on the threshold value. (C) Double mutant cycle free energy differences in the association transition barrier free energy between pairwise I3.6 HCDR3 and CH848.d949 epitope residues. Error bars indicate the standard error of the mean. Errors were propagated from sensorgram fit and technical replicates (n=3) for each measurement set used in calculations. (D) Representative simulation depicting the transition from a near-bound to a prebound state. The root mean square deviation (RMSD; red) is plotted on the left y-axis. The distance between HCDR3 residue D107 γ-carbon and K327 ϵ-amine is plotted on the right y-axis. The pink dashed line indicates the 48.5 ns timepoint at which the stable D107-K327 interaction forms. (E) (left) Near-bound encounter state structure from the representative transition in (D) at the 48.5 ns timepoint. The pink arrow identified the D107 interaction with K327. (right) Pre-bound encounter state structure from the final trajectory state at the 250 ns timepoint. The pink arrow identifies the absence of an interaction between D107 and K327. (F) Double mutant cycle free energy differences in the dissociation transition barrier free energy between pairwise I3.6 HCDR3 and CH848.d949 epitope residues. Error bars indicate the standard error of the mean. Errors were propagated from sensorgram fit and technical replicates (n=3) for each measurement set used in calculations. (G) The DH270 bound state structure (PDB ID 8SB1). The pink arrow identifies the absence of an interaction between D107 and K327.