# Molecular Insights into the Interactions Between Human Serum Albumin and Phospholipid Membranes
## 3 Results and Discussion
### The Affinity of HSA for DPPC and DPPE in the Presence of Mono- and Divalent Cations
Nevertheless, domain III readily interacts with dissolved fatty acids [93; 94; 95], suggesting an affinity of this fragment for negatively charged oxygen atoms, which are also present in the phospholipid headgroups. Indeed, as clearly shown in Figure 3C,D, oxygen atoms of the phosphate group in DPPE form intermolecular interactions with the protein. The analysis of ionic molecular contacts reveals that no such interactions are formed by amino acids from domains IIIA and IIIB in the case of DPPC, irrespective of the cation type. HSA interacts with DPPC primarily through domains IA, IIA, and IIB. In the case of domain IA, ionic interactions are formed at 29%, 24%, 23%, and 21% when the complexes contain Ca\({}^{2+}\), Mg\({}^{2+}\), K\({}^{+}\), and Na\({}^{+}\) ions, respectively. This indicates that Ca\({}^{2+}\) and Mg\({}^{2+}\) cations form slightly more ionic contacts with domain IA than K\({}^{+}\) and Na\({}^{+}\) cations. For domain IIA, the highest number of interactions was identified: 46% (Ca\({}^{2+}\)), 41% (Mg\({}^{2+}\)), 53% (K\({}^{+}\)), and 43% (Na\({}^{+}\)). Interestingly, the presence of K\({}^{+}\) cations contributes to the greatest number of interactions involving DPPC. In this case, domain IIB shows no clear preference for cations in terms of the number of ionic contacts formed. This part of albumin is characterized by a negligible number of ionic contacts (1% regardless of the ions contributing to stabilizing the given complex). In the case of DPPE complexes, ionic interactions were identified in all HSA domains. The highest number of such molecular contacts was identified for domain IA: 29% (Ca\({}^{2+}\)), 32% (Mg\({}^{2+}\)), 33% (K\({}^{+}\)), and 32% (Na\({}^{+}\)), and the lowest for domains IB: 10% (Ca\({}^{2+}\)), 9% (Mg\({}^{2+}\)), 10% (K\({}^{+}\)), and 9% (Na\({}^{+}\)) and IIIA: 12% (Ca\({}^{2+}\)), 9% (Mg\({}^{2+}\)), and 8% for the complexes stabilized by Na\({}^{+}\) and K\({}^{+}\). Domain IIIB, in most cases except for the complexes in the presence of K\({}^{+}\), is characterized by a higher percentage of ionic interactions: 16% (Ca\({}^{2+}\)), 12% (Mg\({}^{2+}\)), 12% (K\({}^{+}\)), and 8% (Na\({}^{+}\)). Domain IIA forms fewer interactions than domain IA. For domain IIA, most interactions with DPPE occur in the presence of K\({}^{+}\) and Na\({}^{+}\) (22% and 23%, respectively). The presence of divalent cations causes the formation of slightly fewer ionic contacts: 19% (Ca\({}^{2+}\)) and 20% (Mg\({}^{2+}\)). Similar to previously discussed ionic interactions, DPPE forms more hydrogen bonds with HSA than DPPC (Figure 7). However, this difference is less pronounced. It is worth noting that the methylated ammonium group in DPPC is completely blocked from forming hydrogen bonds. Consequently, hydrogen bonds in DPPC are exclusively formed with oxygen atoms in the phosphate group acting as acceptors. In contrast, DPPE's amine groups can engage in hydrogen bonding with the oxygen atom of the ASP residue (d = 1.87 \(\pm\) 0.15 A, Figure 3D). This, in turn, results in enhanced stability of HSA-DPPE complexes relative to those formed with DPPC (Figure 3B). The contributions of specific HSA domains to hydrogen bond formation are detailed in Table S2 (Supplementary Materials). In complexes formed by DPPC, only IA, IB, and IIB domains participate in hydrogen bonding. Notably, for DPPC/HSA/Mg\({}^{2+}\) systems, no hydrogen bond interactions with the IB subdomain are detected. In the case of Na\({}^{+}\), K\({}^{+}\), and Ca\({}^{2+}\), the IB subdomain forms a negligibly small number of intermolecular contacts with DPPC. In contrast, in DPPC/HSA/K\({}^{+}\) complexes, hydrogen bonds involving domain IIA contribute significantly, exceeding 50% of the total interactions. Interestingly, ionic interactions involving domain IIA also account for more than 50% of the total interactions (Table S1, Supplementary Materials). Moreover, in the majority of DPPC complexes, domain IIA likewise forms most of the hydrogen bonds and ionic contacts.