4E10 and PGZL1 CDR lipid phosphate binding sites and membrane interaction profiles in all- atom fluid HIV-like bilayer MD simulations

(A) Representative frame from an MD simulation of the phospholipid complex at 4E10 Fab CDR-H1 (top left). Top right, time-averaged lipid phosphate density (orange mesh) relative to antibody CDR loops embedded in the bilayer (beige) from 4 µs total time (top right). Center right, CDR-H1 loop side chain and backbone atoms of de novo predicted phosphate interaction (middle right). Bottom, putative lipid phosphate binding site observed in an X-ray structure for 4E10 (PDB: 4XC1), comparing the CDR-H1 loop interactions to MD (bottom right).

(B) PGZL1 lipid phosphate interaction at the CDR-H1 loop from MD simulation versus X-ray crystallography (PDB: 6O3J), demarked as in (A).

(C) RMSD of the interacting lipid phosphate versus the experimental CDR-phosphate position (X-ray site), classified as bound (green) and unbound (grey) by loop-phosphate contacts in 2 4E10 representative replicate trajectories. Black line, ten-frame RMSD running average; standard deviation, grey shading.

(D) RMSD of lipid phosphate binding to PGZL1 CDR-H1 in MD simulations versus X-ray site.

(E) Per-residue interaction profiles for Fab simulations of 4E10 detailing the time spent for each residue in in phosphate layer (orange), glycerol layer (red), or hydrocarbon layer (blue) across aggregate 4 µs from 4 simulations. CDR loops are mapped in solid color blocks below each profile, and Fab domain regions making significant contact are labeled.

(F) Per-residue interaction profiles for antibody Fab simulations for PGZL1, colored as in (E).

10E8 bivalent lipid headgroup interaction and bilayer insertion predicted in MD simulation closely matches experimental lipid binding sites

(A) Top left, representative frame from MD simulation of lipid interacting with 10E8 Fab. Top right, the MD lipid binding site includes a bivalent choline and phosphate lipid headgroup complex (represented as blue and orange mesh time-averaged positional density in simulations) within a protein surface groove composed of CDR-L1 and FR-L3 respectively. Bottom, positions of phosphates or glycerols modeled at the CDR-L1 and FR-L3 groove site within 10E8 Fab X-ray structures (PDB: 5T85, 5T6L)

(B) RMSD of lipid choline position in the MD simulations versus expected CDR-L1 lipid binding site from 10E8 X-ray structures. “Bound” state assigned relative to choline position and phosphate FR-L3 interactions observed in MD.

(C) Per-residue interaction profiles for antibody Fab simulations for 10E8, colored as in Figure 1E.

Atomistic simulations of apo and antigen-bound LN01 characterizing paratope-phospholipid complexes with and without epitope and shifted membrane-bound conformation

(A) Representative frame from MD simulation LN01 Fab bound to MPER-TM showing the stable ternary paratope-epitope-membrane complex; bound phospholipids shown.

(B) Frequency of Fab’s characteristic surface-bound geometry by global domain rotation and approach angles in MD simulations for LN01 bound to MPER-TM, plotted by kernel density estimation as contour.

(C) Representative frame from MD simulation of phospholipids complexed with LN01 Fab alone.

(D) Frequency of geometries sampled for apo membrane-bound LN01.

(E) Phospholipid headgroup interaction formed ab initio in LN01+MPER-TM simulations. Aromatic cation-pi cage motifs coordinate choline while the phosphate is coordinating by Lys31 matching the X- ray site binding pose.

(F) The additional distal “Loading” phospholipid site predicted in LN01simulations, with a similar cation-pi cage motif and hydrogen bonds interactions stabilizing the PC headgroup.

(G) Atomic interactions at the X-ray site in apo LN01 simulations.

(H) Interactions at the loading site in apo LN01 simulations.

(I) Lipid headgroup binding in representative simulation of LN01+MPER-TM (n=4 total). Top, X-ray binding site occupancy (green) and phospholipid choline RMSD in a representative trajectory versus experimental position. Bottom, loading site occupancy (cyan) and choline RMSD versus average headgroup bound position.

(J) Lipid headgroup binding for representative apo LN01 trajectory (n=4 total). Site occupancy and RMSD versus predicted binding position for X-ray site (top) and loading site (bottom).

(K) Per-residue interaction profile for MPER-TM-bound LN01.

(L) Per-residue interaction profile for apo LN01.

Unbiased spontaneous membrane insertion events and semi-biased dissociation events in coarse grain MD simulations

(A) Snapshots of spontaneous insertion event from a Martini model coarse-grain simulation of a 4E10 Fab. The Fab begins in explicit water solvent 1-2 nm above a lipid bilayer, freely diffusing and tumbling in bulk solvent, often resulting in a temporary or permanent insertion event (right).

(B,C,D) 18 replicates of coarse grain Fab systems (4E10, PGZL1, 10E8, respectively), initialized with slightly different Fab orientations relative to lipid bilayer. Frames with Fab contacting the membrane are in green and frames with Fab in water (non-associated) are in white for replicate trajectories of 14 µs each.

(E) 18 replicates of coarse grain BSA (top) or 13h11 (bottom) with different starting orientations relative to the lipid bilayer. Membrane contact (green) or diffusion in water (white) shown over 10 µs time.

(F) Snapshots describing a co-assembling membrane pipeline with 4E10 Fab. An Fab is centered in a box with various rotational orientations in space, explicit water, and lipids randomly arranged within a subset of the box (left). By 30ns, the membrane is fully formed (middle). Fab molecules result in a pre-docked membrane bound conformation and sample a permanent insertion event, intermittent membrane association, or dissociation depending on how the Fab contacts with the membrane (right).

(G, H, I) 40 replicates of 5 µs simulations for coarse grain co-assembling systems (4E10, PGZL1, 10E8, respectively), each with slightly different Fab initial orientations relative to lipid bilayer. Membrane contact is classified as above.

Membrane surface-bound bnAb conformations sampled across multiscale simulations

(A) Graphic of angles defined to describe Fab geometries relative to the normal vector at the membrane’s upper leaflet lipid at the phosphate plane in simulation frames (orange arrow). The canonical “approach angle” defines the long axis of the Fab domain (i.e. the central pseudo-symmetry axis) and membrane normal vector (black arrow). A second “rotational angle” is defines the global domain rotation about the Fab pseudo-symmetry axis relative to the membrane normal vector, based on the short axis traversing the light and heavy chains, which is nearly orthogonal to the Fab’s central axis (red arrow).

(B) Frequency plots of rotation and approach angles from frames of membrane-bound Fabs in MD simulations for 4E10 (blue, top row), PGZL1 (red, middle row), and 10E8 (purple, bottom row). Contour plots depicting frequency maxima for angle pairs sampled are by kernel density estimation. Left column, membrane interaction angles sampled from all-atom simulations with Fabs pre-docked using OPM PPM server prediction. Middle column, geometries from coarse-grain membrane co-assembly simulations. Right column, geometries from unbiased spontaneous insertion coarse-grain simulations. Black dots denote the initial Fab-membrane geometries of starting states for replicate trajectories for each antibody initiated in the lipid bilayer.

Back-mapping CG membrane-bound geometries to all-atom simulations allows integrative ab initio modeling of the full bnAb insertion process.

(A) Representative frames from membrane-bound 4E10 Fab coarse-grained simulations were back-mapped to all-atom representation to assess the stability and plausibility of those membrane-bound conformations. This coarse-grained-to-all-atom (CG-to-AA) reversion was applied for all medoid frames of interest from each antibody system and used to initiate half-microsecond unbiased all-atom dynamics simulations.

(B) Frequency of membrane interaction angles from coarse grain spontaneous insertion as clustered by geometric substates for 4E10, PGZL1 and 10E8, colored and contoured as in Fig 3B. Corresponding primary all-atom simulations is overlaid (unfilled, black contour frequency density plot).

(C) Conformational geometry sampled upon conversion of CG medoid to an all-atom trajectory. Initial geometry denoted by stars colored matching CG clusters in (B). Frequency and contour plots of conformational angles sampled in stably inserted backmapped all-atom trajectories for 4E10 (left), PGZL1 (middle), and 10E8 (right).

(D) Per-residue interaction profiles for antibody Fab simulations for 4E10 (left), PGZL1 (middle), 10E8 (right) representing each backmapped atomic trajectory, showing CDR-mediated conformations of differing depths and geometries.

(E) Phospholipid headgroup binding and RMSD plots of closest lipid at respective experimentally determined CDR sites for 4E10 (left), PGZL1 (middle), and 10E8 (right), plotted as in Figure 1C or 2B.

Biased antibody-membrane pulling dissociation simulations approximate Fab-membrane interaction strength for bnAb Fab variants of known lipid binding affinity or neutralization potency.

(A) Schematic of pulling method, a bnAbs Fabs associated with the lipid bilayers is subject to an applied upward dissociation force to the Fab domain center of mass at constant velocity to measure the force required to dissociate the Fab from the bilayer.

(B) The Trp100a-Trp100c motif residues in 4E10 CDR-H3 loop of expected to be lipid-embedded in the bilayer (beige). The double alanine mutant “WAWA” 4E10 variant has experimentally determined significantly reduced affinity to lipid bilayers and lower neutralization potency, due to lack of Trp membrane insertion.

(C) Previously experimentally characterized20 PGZL1 germline-reverted variants, shown as chimera of germline versus matured gene segments, used to approximate antibody properties along its maturation trajectory.

(D) Average force versus distance plots and rupture force (Fmax) calculation for one replicate of pulling wild type 4E10 (WT, blue) and 4E10 WAWA (red) to bias Fab dissociation from the bilayer.

(E) Distribution of rupture forces required for dissociation for different membrane-bound starting conformations for 4E10 (n=9, blue) and 4E10 WAWA (n=11, red), with starting conformations drawn from previous unbiased all-atom simulation ensembles at rest. Outliers as dots

(F) Distributions of rupture forces (n=10) required for membrane dissociation of PGZL1 inferred variants along the maturation pathway, for germline (dark grey, Grm.), intermediate (light grey, Int.), and mature (maroon, Mat.) PGZL1. Outliers as dots