Unraveling the Molecular Dance: Insights into TREM2/DAP12 Complex Formation in Alzheimer’s Disease through Molecular Dynamics Simulations

Alzheimer’s disease (AD) is a widespread neurodegenerative condition affecting millions globally. Recent research has implicated variants of the triggering receptor expressed in myeloid cells 2 (TREM2) as risk factors for AD. TREM2, an immunomodulatory receptor on microglial surfaces, plays a pivotal role in regulating microglial activation by association with DNAX-activation protein 12 (DAP12). Despite its significance, the mechanism underlying the formation of the complex between the transmembrane domains (TMDs) of TREM2 and DAP12 remains unclear. This study employs multiscale molecular dynamics (MD) simulations to investigate three TMD complex models, including two derived from experiments and one generated by AlphaFold2. Conducted within a lipid membrane consisting of an 80:20 mixture of phosphatidylcholine (POPC) and cholesterol, our analysis reveals hydrogen-bonding interactions between K26 of TREM2 and D16 of DAP12 in all three models, consistent with previous experimental findings. Our results elucidate the different spatial conformations observed in the models and offer insights into the structure of the TREM2/DAP12 TMD complex. Furthermore, we elucidate the role of charged residues in the assembly structure of the complex within the lipid membrane. These findings enhance our understanding of the molecular mechanism governing TREM2/DAP12 complex formation, providing a foundation for designing novel therapeutic strategies to address AD and other neurodegenerative diseases.


Supporting Information Text
Stability check of the three systems.Figure S1 depicts the total potential contacts between TREM2 and DAP12.The data spans four different timescales (25 -75 ns, 100 -150 ns, 175 -225 ns, and 250 -300 ns) within a 300 ns all-atom (AA) simulation, comprising 50 ns simulation intervals.Potential contacts are assessed based on C α atom distances between TREM2 and DAP12 that are less than 11.0 Å.In the case of 6z0g TD, the potential contacts for Chain A occur between 100 -150 ns, while those for Chain B span 70 -150 ns.For 6z0i TD, the potential contacts for Chain A remain relatively stable throughout the simulation, reaching around 100 at the conclusion.In contrast, the potential contacts for Chain B fluctuate significantly between 25 -75 ns and stabilize toward the end of the simulation, also reaching a count of about 100.In the case of AF TD, the potential contacts exhibit a slight decrease and eventually reach about 100 in both chains.We used the potential contact maps to assess the stability of the three systems.In the 6z0g TD system (Figure S2), the potential contact maps for both Chain A (Figure S2A) and Chain B (Figure S2B) of DAP12 exhibit a high degree of similarity, suggesting a stable state in the complex.
In the 6z0i TD system, the potential contact maps for Chain A (Figure S3A) of DAP12 are largely similar.However, minor differences are noticed in the 175 -225 ns period for 6z0o TD Chain B (Figure S3B).These discrepancies are observed in the N-terminal regions of the TREM2/DAP12 trimer, known for containing unstructured loops.Our focus on the α-helix region indicates acceptable consistency in the middle region contact maps across all three contact maps.
As for the AF TD system, the potential contact maps exhibit consistency across the 25 -75 ns, 100 -150 ns, and 175 -225 ns periods (Figure S4).However, in the 250 -300 ns period, a decrease in contacts is noted in both chains within the N-terminal region of TREM2/DAP12, while the middle region interactions remain stable.These observations indicate a stable interaction within the TREM2 and DAP12 transmembrane domain (TMD) post the coarse-grained simulations.
Confidence of the TREM2/DAP12 complex generated by AlphaFold2.Fig S5 shows the confidence of the AlplaFold2 predicted structures.
Contact maps of TREM2/DAP12 complex.Figure S6 illustrates the contact maps of TREM2/DAP12 using a cutoff distance of 3 Å for all atoms, including side chain atoms.
Meanwhile, Figure S7 depicts the regenerated potential contact maps of the TREM2/DAP12 complex.It uses an indexing system where a dot is placed in the contact map if the contact time exceeds 50 % of the total time.
Detailed hydrogen bond possibilities of the TREM2/DAP12.Table S1 -S3 shows the hydrogen bond possibilities.
Mergerd hydrogen bond interaction.Figure S16 shows the merged hydrogen bond interaction.
Hydrophobic interaction of key residues.Figure S17, S18, S19, and S20 display the residue pairs involved in hydrophobic interactions, namely L19/I12, W23/I23, W34/A24, and W34/V27, respectively.Figure S21 illustrates the distance between these residue pairs to determine hydrophobic interactions.In this analysis, a hydrophobic interaction is defined when the distance between any carbon atoms within a particular residue and another residue is below 5 Å. Figure S22 shows the facing direction of hydrophobic residues in three systems.
The snapshots were captured in 150 ns.
Sequence of three systems.Table S4 shows the amino acid sequence of our built systems.
Stability check of the three systems.We used the potential contact maps to assess the stability of the three systems.In the 6z0g TD system (Figure S2), the potential contact maps for both Chain A (Figure S2A) and Chain B (Figure S2B) of DAP12 exhibit a high degree of similarity, suggesting a stable state in the complex.
In the 6z0i TD system, the potential contact maps for Chain A (Figure S3A) of DAP12 are largely similar.However, minor differences are noticed in the 175-225 ns period for 6z0o TD Chain B (Figure S3B).These discrepancies are observed in the N-terminal regions of the TREM2/DAP12 trimer, known for containing unstructured loops.Our focus on the α-helix region indicates acceptable consistency in the middle region contact maps across all three contact maps.
As for the AF TD system, the potential contact maps exhibit consistency across the 25 -75 ns, 100 -150 ns, and 175 -225 ns periods (Figure S4).However, in the 250 -300 ns period, a decrease in contacts is noted in both chains within the N-terminal region of TREM2/DAP12, while the middle region interactions remain stable.These observations indicate a stable interaction within the TREM2 and DAP12 transmembrane domain (TMD) post the coarse-grained simulations.S3: Hydrogen bonds possibility of W34/T20. Figure S17, S18, S19, and S20 display the residue pairs involved in hydrophobic interactions, namely L19/I12, W23/I23, W34/A24, and W34/V27, respectively.Figure S21 illustrates the distance between these residue pairs to determine hydrophobic interactions.

AlphaFold2 predicted structures
In this analysis, a hydrophobic interaction is defined when the distance between any carbon atoms within a particular residue and another residue is below 5 Å.
Figure S1 depicts the total potential contacts between TREM2 and DAP12.The data spans four different timescales (25 -75 ns, 100 -150 ns, 175 -225 ns, and 250 -300 ns) within a 300 ns all-atom (AA) simulation, comprising 50 ns simulation intervals.Potential contacts are assessed based on C-α atom distances between TREM2 and DAP12 that are less than 11.0 Å.In the case of 6z0g TD, the potential contacts for TD Chain A occur between 100 -150 ns, while those for TD Chain B span 70 -150 ns.For 6z0i TD, the potential contacts for Chain A remain relatively stable throughout the simulation, reaching around 100 at the conclusion.In contrast, the potential contacts for Chain B fluctuate significantly between 25 -75 ns and stabilize toward the end of the simulation, also reaching a count of about 100.In the case of AF TD, the potential contacts exhibit a slight decrease and eventually reach about 100 in both chains.

Figure S1 :
Figure S1: Total contacts across the three systems.Distinctly coloured rectangles indicate various time scales: green, blue, red, and yellow respectively represent 25 -75 ns, 100 -150 ns, 175 -225 ns, and 250 -300 ns.Contacts are colour-labelled according to the different chains within DAP12; sea-green designates the interaction of TREM2 and DAP12 Chain A, while orange represents the interaction of TREM2 and DAP12 Chain B.

Figure S2 :
Figure S2: 3D structure snapshots and potential contact maps of 6z0g TD. (A) The four 3D structures were captured at different times (50 ns, 125 ns, 200 ns, 275 ns) and are represented by differently coloured rectangles, consistent with Figure S1.The blue-coloured α-helix represents TREM2, while the green-coloured α-helix shows DAP12 Chain A. The orange sphere denotes the C-α atom of K26 in TREM2.The potential contact maps depict normalized contact numbers, with black indicating 0 and white indicating 1.These contact maps were calculated over four different timescales (25 -75 ns, 100 -150 ns, 175 -225 ns, and 250 -300 ns), consistent with Figure S1.(B) In the 3D structure screenshots, the bluecoloured α-helix represents TREM2, while the red-coloured α-helix shows DAP12 Chain B. The contact maps were normalized to 0 and 1, represented by deep blue and yellow, respectively.

Figure S3 :
Figure S3: 3D structure screenshots and potential contact maps of 6z0i TD. (A) and (B) are consistent with Figure S2.

Figure S4 :
Figure S4: 3D structure screenshots and potential contact maps of AF TD. (A) and (B) are consistent with Figure S2.

Figure S5 :
Figure S5: Confidence of AlphaFold2 predicted structures.(A) PAE plots featuring two domains with low conformation confidence.Five different models are run, rank 1 to rank 5 respectively.(B) The multiple sequence alignment is summarised as a heatmap.The heat map representation of the MSA indicates all sequences mapped to the input sequences.The colour scale indicates the identity score, and sequences are ordered from top (largest identity) to bottom (lowest identity).White regions are not covered, which occurs with sub-sequence entries in the database.The black line qualifies the relative coverage of the sequence with respect to the total number of aligned sequences.(C) The predicted IDDT (I-Domain Distance Test)) per residue for the 5 models was obtained after an AlphaFold2 job.(D) The predicted 3D structure of AlphaFold2 is coloured in predicted LDDT (Local Distance Difference Test).

Figure S6 :
Figure S6: Contact maps of TREM2/DAP12 complex.(A) The contact maps of the 6z0g TD system between TREM2 and DAP12 Chain A or Chain B (left and right, respectively).(B) The contact maps of 6z0i TD system between TREM2 and DAP12 Chain A or Chain B (left and right, respectively).(C) The contact maps of the AF TD system between TREM2 and DAP12 Chain A or Chain B (left and right, respectively).

Figure S7 :
Figure S7: Revised potential contact maps.These calculated potential contact maps were generated using 300 ns trajectory data, and the distance between Cα atoms was below 11 Å for no less than 90% of the whole simulation time.(A) shows the calculated contact maps of 6z0g TD.The contacts between DAP12 Chain A and TREM2 are in green, and between DAP12 Chain B and TREM2 are in orange.The red rectangle represents the α-helix, and residues in the rectangle indicate the residues in the α-helix, while those outside are in the loop region.(B) shows the calculated contact maps of 6z0i TD. (C) shows the calculated contact maps of AF TD.

Figure S8 :
Figure S8: Contact maps of L19/I12 residues.(A) The contact maps of L19/I12 residues in 6z0g TD system.(B) The contact maps of L19/I12 residues in 6z0i TD system.(C) The contact maps of L19/I12 residues in AF TD system.

Figure S9 :
Figure S9: Contact maps of K26/D16 residues.(A) The contact maps of K26/D16 residues in 6z0g TD system.(B) The contact maps of K26/D16 residues in 6z0i TD system.(C) The contact maps of K26/D16 residues in AF TD system.

Figure S10 :
Figure S10: Contact maps of K26/T20 residues.(A) The contact maps of K26/T20 residues in 6z0g TD system.(B) The contact maps of K26/T20 residues in 6z0i TD system.(C) The contact maps of K26/T20 residues in AF TD system.

Figure S11 :
Figure S11: Contact maps of W34/T20 residues.(A) The contact maps of W34/T20 residues in 6z0g TD system.(B) The contact maps of W34/T20 residues in 6z0i TD system.(C) The contact maps of W34/T20 residues in AF TD system.

Figure S12 :
Figure S12: Contact maps of W34/I23 residues.(A) The contact maps of W34/I23 residues in 6z0g TD system.(B) The contact maps of W34/I23 residues in 6z0i TD system.(C) The contact maps of W34/I23 residues in AF TD system.

Figure S13 :
Figure S13: Contact maps of W34/A24 residues.(A) The contact maps of W34/A24 residues in 6z0g TD system.(B) The contact maps of W34/A24 residues in 6z0i TD system.(C) The contact maps of W34/A24 residues in AF TD system.

Figure S14 :
Figure S14: Contact maps of W34/V27 residues.(A) The contact maps of W34/V27 residues in 6z0g TD system.(B) The contact maps of W34/V27 residues in 6z0i TD system.(C) The contact maps of W34/V27 residues in AF TD system.

Figure S15 :
Figure S15: Contact maps of W34/Y28 residues.(A) The contact maps of W34/Y28 residues in 6z0g TD system.(B) The contact maps of W34/Y28 residues in 6z0i TD system.(C) The contact maps of W34/Y28 residues in AF TD system.

Figure S16 :
Figure S16: Residues distance of three systems.

Figure S17 :
Figure S17: Hydrophobic interaction of L19/I12 in three systems.(A) The hydrophobic interaction of L19/I12 residues in 6z0g TD system.(B) The hydrophobic interaction of L19/I12 residues in 6z0i TD system.(C) The hydrophobic interaction of L19/I12 residues in AF TD system.

Figure S18 :
Figure S18: Hydrophobic interaction of W23/I23 in three systems.(A) The hydrophobic interaction of W23/I23 residues in 6z0g TD system.(B) The hydrophobic interaction of W23/I23 residues in 6z0i TD system.(C) The hydrophobic interaction of W23/I23 residues in AF TD system.

Figure S19 :
Figure S19: Hydrophobic interaction of W34/A24 in three systems.(A) The hydrophobic interaction of W34/A24 residues in 6z0g TD system.(B) The hydrophobic interaction of W34/A24 residues in 6z0i TD system.(C) The hydrophobic interaction of W34/A24 residues in AF TD system.

Figure S20 :
Figure S20: Hydrophobic interaction of W34/V27 in three systems.(A) The hydrophobic interaction of W34/V27 residues in 6z0g TD system.(B) The hydrophobic interaction of W34/V27 residues in 6z0i TD system.(C) The hydrophobic interaction of W34/V27 residues in AF TD system.

Figure S21 :
Figure S21: Hydrophobic interaction of key residues in three systems.(A) The hydrophobic interaction of L19/I12 residues in three systems.(B) The hydrophobic interaction of W34/I23 residues in three systems.(C) The hydrophobic interaction of W34/A24 residues in three systems.(D) The hydrophobic interaction of W34/V27 residues in three systems.

Figure S23 :
Figure S23: Contact maps of different time frames of CG simulation.(A) Contact maps of 6z0i TD in 0-50 us.Top map showing the contacts of TREM2 and DAP12 chain A, and the bottom map showing the contacts of TREM2 and DAP12 chain B. (B) Contact maps of 6z0i TD in 50-100 us.(C) Contact maps of 6z0i TD in 150-200 us.

Table S1 :
Hydrogen bonds possibility of K26/D16.A is refered to Chain A. b B is refered to Chain B. a