Human Serum Albumin (PDB ID: 2BXN)
Created by Daniel Loriaux
Hydrophobic substrate molecules are transported in human plasma and extravascular fluids via carrier proteins, and human serum albumin is the most abundant protein in the circulatory system. Human serum albumin comprises approximately 60% of the plasma protein and the versatile binding properties of HSA make it a major contributor to the oncotic pressure of blood (4,6).
Human serum albumin (HSA, 2BXN) consists of a single chain, 585 amino acids in length, which incorporates
three homologous domains (I, II, and III). Domain I consists of residues 5-197, domain II includes residues 198-382, and domain III is formed from residues 383-569. Each domain is comprised of two
sub-domains termed A and B (IA; residues 5-107, IIA; residues 108-197, IIA; residues 198-296, IIB; residues 297-382, IIIA; residues 383-494, IIIB; residues 495-569) (3). HSA regulates colloidal osmotic pressure of the blood via its capacity to bind to fatty acids, hormones, pharmaceuticals, and bilirubin (a drug-site 1 binding ligand)7. Bilirubin is able to complex with HSA at a single high-affinity binding site fashioned from
residues 180-250 of albumin (9). HSA, PDB entry 2BXN, includes iodipamide and myristic acid as its two representative ligands. Iodipamide has been shown to coordinate to drug binding site 1 by inducing a rotation of the W-214 side-chain (9). Of the 585 amino acids that comprise HSA (2BXN), W-214 presents the greatest contact area (57%). HSA utilizes a combination of elecrostatic interactions, hydrogen bond interactions, and van der Waals interactions to bind up to six molecules of myristic acid and two molecules of iodipamide. The residues that enable HSA's interaction with iodipamide include L-198, L-481, K-199, F-211, A-291, L-238, R-257, A-291, R-218, and W-214 at
iodipamide binding site 1, and K-432, W-452, D-187, A-191, and E-188 at
iodipamide binding site 2 (15). Although HSA integrates three homologous domains, the
myristate binding properties of each domain are quite unique (5). The first equivalent,
Myr 1, is accommodated within a pocket on sub-domain IB (residues 5-107),
Myr 2 binds to a cavity formed principally by the interface of IA and IIA (residues 5-107 and 205-296),
Myr 3 binds to sub-domain IIIA (residues 383-494) and the Myr 3 carboxylate moiety is anchored by a portion of sub-domain IIB,
Myr 4 binds to sub-domain IIIA (residues 383-494),
Myr 5 binds to a similar site as Myr 1 on the homologous sub-domain IIIB (resides 495-569) but displays an inverted orientation relative to Myr 1, and
Myr 6 binds by via the residues L-347, R-209, and D-3245.
In its biologically active state, HSA is an extremely versatile monomer with flexible structural organization (evident in the 71% helical structure). Although predominantly alpha-helical, other
secondary structures include 28 beta-turns, 2 gamma-turns, and 17 disulfide bridges. Cooperativity and allosteric modulation characterize the binding sites of HSA and allow HSA to functionally mimic the activity of a multimeric enzyme. Localized defects in protein packing often culminate in closed cavities, which can potentially reduce the stability of a protein's structure. Beyond their classification as "packing defects," however, closed cavities are also credited as facilitators of conformational changes within the protein's monomeric structure (14). There are 19 total
closed cavities inherent in the HSA structure. Problems with adsorption, distribution, metabolism, and elimination must be considered and addressed throughout the development of any new drug. Thus, the biological function of human serum albumin is of great interest interest to pharmaceutical companies because HSA is an instrumental protein in dictating the active concentrations of administered drugs. HSA's two drug-binding sites are studded with basic side chains, thus conferring an affinity for anionic, lipophilic drugs.
HSA possesses two binding pockets that are designated as specialized drug binding sites. These two pockets are found within the core of subdomains IIA and IIIA1. Non-esterified fatty acids are the primary physiological ligands of HSA. and the predominantly apolar nature of HSA's binding pockets promotes van der Waals interactions between HSA and the associated fatty acid substrate. Human serum albumin achieves substrate selectivity via a small class of basic residues that are positioned both posteriorly
(W-150, H-242, R-257) and around the periphery of drug binding site 1
(K-195, K-199, R-218, R-222) (5). Through these residues, HSA effectively anchors the substrate to the binding site via hydrogen bond interactions. Experimental data suggests that the majority of HSA drug site 1/substrate interactions involve K-199, R-222, and H-242. The existence of basic, positively charged residues and the absence of glutamate and aspartate residues is a distinctive structural feature of subdomian IIA that is emblematic of the specificity of function demonstrated by this binding site. As will be discussed later, subdomain IIA plays a crucial role in the conformational change that HSA is able to undergo. During the conformational change that is possible upon the binding of long-chain fatty acids, two subdomain IIIA acidic residues, ( E-450 and D-451), both rotate to new positions (5). E-450 displaces D-451, which enables D-451 to relocate and form a salt bridge with K-195 (3,5). Reagents targeting site 1 are generally anionic or electronegative species that are inherently attracted to the positively charged residues positioned within the pocket of subdomain IIA1. Although electrostatic interactions between the drug substrate and HSA account for a large portion of the substrate's affinity for the albumin IIA binding site, hydrogen bond interactions and Van-der Waals forces also contribute to the overall attraction and specificity of the site 7,8,10. Every compound that complexes with
drug binding site 1 in subdomain IIA orients itself so as to generate a hydrogen bond interaction with W-150, making this residue essential for proper enzymatic function and drug transportation. If W-150 is occupied in a hydrogen bond with the carboxylate moiety of a fatty-acid then the IIA site is no longer capable of the drug-binding interaction that is observed with defatted HSA. Although the interaction with W-150 is sacrificed upon fatty acid binding, the interaction with H-242 is retained1,3.
Drug site 2, located in subdomain IIIA, is topologically similar to site 1. Site 2 mimics site 1 in its hydrophobic nature and its polarity; it is composed of a large hydrophobic cavity with polar features once again contributed by basic residues. Site 2 differs from site 1 in size and accessibility. Site 2 is smaller with an entrance that is less sterically hindered and more exposed to the surrounding environment. In contrast to site 1, site 2 bears only a single polar patch that is constricted to one side of the pocket and centered on W-411, R410, K-414, and S-489 (4). Of these residues, only R-410 and K-414 are conserved in site 1 (R-218 and R-222)1. Drug site 2 involves the hydroxyl group of W-411 as the main interaction partner for drug binding, and a published crystal structure of
ibuprofen complexed with defatted HSA (PDB 2BXG) shows that binding of ibuprofen in this site primarily involves interactions with R-410, W-411, and S-4896. Drug-site 2, like drug-site 1, associates with the fatty acid substrate via a predominantly apolar pocket that interacts with the methylene tail of the fatty acid while the polar periphery of the pocket interacts with the carboxylate moiety of the substrate (1). K-351, S-480, L-481, and V-482 are residues contributed by subdomains IIB (K-351) and IIIA (S-480, L-481, V-482) that comprise a critical binding region in drug site 2. These residues influence pharmacokinetics and pharmacodynamics by interacting with the carboxylate moiety of the drug substrate (1). The discrepancies that exist between the HSA drug-binding sites are sufficient for sites 1 and 2 do be distinguishable from one another in terms of polarity, size, and shape. These differences help to explain the unique binding specificities and substrate selectivity of each site.
Vitamin-D-binding protein (DBP, 1KW2), a homolog of human serum albumin, is a transport protein in the homeostatic actin-scavenger mechanism that regulates the concentration of actin in the blood (2,6). DBP resembles albmumin from a functional perspective in that it fulfills a similarly vital binding/transport role in the circulatory system. Although actin is the most prominent protein in eukaryotic cells, in the absence of DBP the release of actin into circulation would have lethal consequences. DALI and BLAST searches conclude that DBP has the highest total score (272), query coverage (95%), and E Value (2e-33) of all PDB entries that do not simply interchange the ligands associated to HSA13,14,15. Considering that HSA undergoes the same conformational change upon binding any fatty acid (>C10:0), merely changing the identity of the associated substrate will culminate in a 100% identity observed between the two HSA PDB entries. PDB entry 1GNJ, HSA complexed with arachidonic acid, and PDB entry 2BXN, HSA complexed with myristic acid and iodipamide, can be
superimposed to visually confirm that altering the identity of the fatty acid bound to HSA does not modify the structure. BLAST sequence alignment results indicate that HSA and DBP possess analogous primary stuctures and crystallization data verifies that both of these transport proteins include three fundamental domains. Despite the analogous data, however, the tertiary structures of the two homologs are quite distinct from one another. Although HSA and DBP are close functional homologs,
superimposition of HSA and DBP demonstrates that their structures are not as highly conserved.
Unliganded HSA (PDB = 1AO6), when compared with the HSA-myristate complex (PDB = 2BXN), reveals that HSA undergoes a slight conformational change upon binding to a molecule of myristate substrate; domain I and domain III pivot around domain II and the
continuous helix connecting domains I and II bends when the fatty acid complex is formed.6,8,10. Fatty acid binding site 2 (FA site 2), which traverses subdomains IA and IIA, is implicated as the driving force of the conformational change that ensues from fatty acid binding4. FA site 2 offers the most enclosed environment out of the seven long-chain fatty acid binding sites, and the binding of a fatty acid in site 2 prompts the rotation of domains I and III relative to domain II4,5. Crystallographic analyses suggest that C10:0-C18:0 fatty acids possess methylene tails that are long enough to occupy both subdomains (IA and IIA) of FA site 2, and this explains why it is only long-chain fatty acids that are capable of inducing the HSA conformational change. C8:0 fatty acids are too short to extend from subdomain IIA into subdomain IA and therefore no such conformational change is observed in the shorter-chain fatty acid complexes (4).
The function of HSA remains uniform regardless of whether or not a conformational change has been induced. However, HSA function can be altered via glycosylation. K-525 has been established as the principal site of in vivo non-enzymatic glycosylation of HSA12. Nonglycosylated albumin demonstrates an affinity for bilirubin that is two-fold what is observed for glycosylated albumin (5,7). A ramification that arises from HSA's unusually broad spectrum of ligands is that many of HSA's asymmetrically distrubuted binding sites demonstrate noticeable overlap, and therefore the activity of certain HSA binding sites are dependent on the occupancy of neighboring sites. The two drug-binding sites present in HSA (subdomain IIA and IIIA) overlap extensively with endogenous ligand-binding sites, and therefore these sites often demonstrate cooperativity. Recent crystallographic studies have mapped
7 different long chain fatty acid binding sites (3,6). Of these seven binding sites, mutagenesis studies have identified three sites in domain III that express particularly high substrate affinity (6). Drug site 1 overlaps with
FA site 7 and drug site 2 overlaps with
FA site 3 and
FA site 4. The extent of site overlap explains the competitive interaction that is seen between pharmaceuticals and fatty acids. The high affinity FA sites (
FA site 2,
FA site 4,and
FA site 5) differ from the lower affinity sites (
FA site 1,
FA site 3,
FA site 6, and
FA site 7) by providing an enclosed binding pocket that is able to accommodate the methylene fatty acid tail in a linear conformation (6). FA site 2, as previously discussed, is especially strong in this regard. Human serum albumin was one of the first proteins to be successfully isolated and purified. Thus, albumin was the subject of much of the earliest research that was conducted on protein structure/function. Ironically, the abundant and multifunctional nature of HSA that initially made albumin so apealing to investigators still drives the research that is being performed by pharmaceutical companies today.