Human Serum Albumin
Created by Ayla Benge
Human serum albumin complexed with dodecanoic acid, pdb id 1E7F, is the most common protein found in blood plasma. Its concentration reaches 40 mg/mL within the bloodstream (5). With a primary sequence length of 585 amino acids and a quaternary structure composed of a single subunit, human serum albumin, or HSA, is a relatively small, heart-shaped protein (6, 5). It has a total molecular mass of 66,472.21 Da and an isoelectric point at 5.67 (4). Dodecanoc acid, also known as lauric acid, is one of the many fatty acids that serum albumin binds to and transports throughout the body. Fatty acids are essential for the synthesis of membrane lipids, hormones, and second messengers and are an important source of metabolic energy. Without HSA, these fatty acids do not get transported throughout the blood. HSA also regulates osmotic pressure and acts as a buffer, an ability that arises from its anionic character. In addition, HSA can transport other hydrophobic molecules, such as pharmacological drugs. This can be potentially problematic as the presence of fatty acids can either inhibit or assist the binding of drugs to HSA (5).
The structure of HSA lends the protein its unique abilities. The protein is 70% helical with a few random coils (6). The homologous sub-domains I, II, and III of HSA each have their own A and B sections (1). Each A sub-domain has 6 alpha helices, and each B sub-domain has 4 helices (8). A total of eleven binding sites have been identified (5). Binding sites 1 through 7 bind lauric acid, C12:0. These sites can also bind capric (C10), palmitic (C16), and stearic (C18) acid. C14, myristic acid, is not found in appreciable quantities in human blood plasma, but it too binds to serum albumin, as determined by crystallographic analysis. Binding sites beyond site 7 only interact with smaller fatty acids with less than ten carbons (1).
A pattern emerges when examining HSA's binding sites. The polar carboxylate heads of the fatty acids possess both a hydroxyl group and a carbonyl carbon that can participate in hydrogen bonds. These carboxylate groups interact with the polar and basic side chains of HSA’s residues, becoming anchored to the protein. The nonpolar methylene tail of the fatty acid extends into the hydrophobic core created by the space between the alpha helices, thereby strengthening binding by participating in multiple van der Waals interactions. In general, as the tail length increases, there is a higher affinity for the fatty acids at these binding sites because of increased electrostatic interactions (1). Thus, the serum albumin molecule is able to effectively transport amphiphillic molecules.
Site 1 is located in sub-domain IB. Tyr138 and Tyr161 block the hydrophobic core of site 1 in the absence of fatty acid. When fatty acid is present, these tyrosines rotate and create a hydrophobic clamp with their phenyl rings, effectively trapping the tail of the fatty acid. The carboxylate head then interacts with Arg117, the hydroxyl of Tyr161, and the carbonyl of Leu182 in a hydrogen bond. Larger fatty acids, such as stearic acid, do not bind well here due to their decreased solubility. A curled tail effect is observed for longer fatty acids so that the end of the methylene tail is not in contact with polar solvent (1).
Hydrogen bonds anchor the carboxylate moiety of fatty acids to Tyr150, Arg257, and Ser287 at site 2. Located in sub-domains IA and IIA, site 2 encloses fatty acids more fully than any of the other sites. The uptake of ligand at site 2 drives a conformational change that creates a narrow hydrophobic cavity between the helices of IA and IIA into which the tail of the fatty acid extends (1).
Sites 3 and 4 are located in sub-domain IIIA and are positioned such that the two fatty acids that bind at these sites are in contact at their tails. Site 3 contains a shorter, wider pocket that causes the fatty acid tail to bend as its carboxylate head hydrogen bonds to Ser342 and Arg348 from sub-domain IIB. The helices from sub-domains IIB and IIIA are approximately perpendicular to each other, thus facilitating this hydrogen bond interaction. Site 4 has a long, narrow hydrophobic tunnel from the domain IIIA helices which contains the nonpolar fatty acid tails. The carboxylate moiety hydrogen bonds to Arg410, Tyr411, and Ser489 from IIIA (1).
At site 5, fatty acids extend through a hydrophobic tunnel in sub-domain IIIB. Longer fatty acids fit snugly within the long pocket. Carboxylate heads interact with the side chain of Lys525 in hydrogen bonding and are assisted by Tyr401. Binding site 6 is unlike the other sites; it does not possess identifiable residues with which the carboxylate moiety can hydrogen bond. Located at the surface of sub-domains IIA and IIB, site 6 has a salt-bridge from Arg209 to Asp324 and Glu354 that helps hold the methylene tail within the molecule. Site 7 is a small binding site positioned in sub-domain IIA. Lys199, Arg218, Arg222, His242, and Arg257 are suspected of hydrogen bonding to the head of the fatty acid tail. The fatty acid takes on a curved configuration in order to fit into the small space provided in IIA. Sites 8 and 9 only bind fatty acid molecules with less than 10 carbon atoms due to the small size of the hydrophobic crevice where the fatty acid tail is bound (1).
Serum albumin’s unique structure allows it to bind to drugs as well as fatty acids. In bovine serum albumin, short-chain fatty acids at site 7 prevent N-dansylaziridine from binding. However, long-chain fatty acids do not block N-dansylaziridine, suggesting that only short chain fatty acids bind at site 7 (1). Bovine serum albumin is highly homologous to human serum albumin and therefore the binding of N-dansylaziridine applies to HSA (9). In contrast, fatty acids bound to serum albumin actually increase the affinity of HSA for warfarin, which binds to site 2 in domain II (3). Bilirubin also binds to HSA at IIA. Association rates of bilirubin and HSA are higher when fatty acid is present in small amounts, but lower when fatty acid is present in large amounts. A conformational change is required for HSA to bind to bilirubin, a change that may be inhibited by the presence of fatty acids (7). Diazapam, more commonly known as Valium, and the digitoxins drugs are carried by HSA through the blood. Ibuprofen and AZT, a drug used to fight AIDS, bind to HSA in sub-domain IIIA (9). Esterase’s enzymatic activity involving Tyr411 in sub-domain IIIA is negatively affected by fatty acids binding to HSA (2). HSA is a biologically important protein because of these drug-binding interactions. Without HSA, these drugs would not be distributed throughout the bloodstream.
Human serum albumin bears similarities to the vitamin D binding protein, pdb ID 1J78, found in humans (12). PSI-BLAST is a database that identifies sequence homology between a query sequence and other sequences by assigning an E value. This program revealed that HSA and the vitamin D binding protein, or DBP, have similar sequences, as indicated by a low E-score, 2e-123 (11). The Dali Server examines tertiary structure of one protein and finds other proteins with similar tertiary structures. Z scores measure this similarity, where a value above 2 indicates high similarity. HSA and DBP share a Z score of 19.7, meaning that their tertiary structures resemble each other greatly (10). Although HSA and DBP are structurally similar, the functions of these two proteins differ significantly.
The vitamin D binding protein has a molecular weight of 102,414.76 Da and an isoelectric point at 5.17 (4). This protein has two heteromeric subunits, as compared to serum albumin’s one, with 458 residues. It is 63% helical and has 21 helices total. Like HSA, DBP binds and transports a fatty acids, such as oleic acid. It also binds to the metabolites of vitamin D3 and vitamin D3 itself. (12). DBP also has three domains composed of helical segments and random coils. While domain I on DBP is the most similar to the 10 helices of the HSA domains, domains II and III differ more noticeably. The helices of domain II are interrupted by a random coil, and domain III possesses only 4 helices. This results in a different folding pattern between the two proteins, both overall and locally. Due to this difference in folding, DBP has a more diverse array of functions than does HSA. DBP removes 25-hydroxyvitamin D3, thereby driving vitamin D3 breakdown. DBP attached to 25-hydroxyvitamin D3 is absorbed in the proximal tubulus, preventing vitamin D3 loss in urine. Furthermore, DBP binds to actin and slowly depolymerizes filamentous actin. The delivery of vitamin D3 hormones by DBP assists in bone homeostasis and helps to regulate cell division (13).
Although serum albumin and the vitamin D binding protein have strong sequence homology and visibly similar tertiary structures, there are key differences in their structures and folding patterns that result in vastly different functions. This differentiation between proteins, despite structural and sequential similarities, is essential to life and to the variation of functions required for cell survival.