Alpha-Lactalbumin
Created by Van Anh Do
Alpha-Lactalbumin (1A4V) from Homo Sapiens is a small and acidic protein as evidenced by its molecular weight (14,200 kDa) [10] and isoelectric point (pI= 4.70) [1]. The data are obtained using ExPASy. ExPASy is the SIB Bioinformatics Resource Portal that provides access to scientific databases and tools in different areas of sciences including proteomics, genomics, phylogeny, systems biology, population genetics, and transcriptomics [1].The Alpha-Lactalbumin (α-LA) is one of the two components of lactose synthase. Lactose synthase consists of α-LA and a galactosyltransferase (GT), UDPgalactose:N-acetylglucosamine β-1,4-galactosyltransferase. While GT alone can utilize glucose, the Michaelis constant for this reaction is about 2M [3]. In the presence of α-LA, the Km of this reaction is reduced to the low millimolar range [3]. Therefore, α-LA interacts with GT and modulates its acceptor substrate specificity so that it can catalyze the transfer to glucose.
α-LA has one subunit. The primary sequence of α-LA contains 123 amino acid residues [10]. There are 35 invariant residues in all known α-LAs of different species [6]. Among them, the cysteine residues at 6, 28, 61, 73, 77, 91, 111 and 120 play important structural role. Specifically, Cys-77 and Cys-91 are of interest since they flank the calcium-binding “elbow” and a pair of orthogonal helical segments. Leu-81, Asp-82, Asp-83 and Asp-87 are located in the calcium-binding “elbow” of α-LA while Lys-1, Asp-37, Glu-39, His-32 and Gly-35 are found in the contact region with GT in the lactose synthase complex. In fact, His-32 and Trp-118 may be essential for lactose synthase activity [6].
The native secondary structure of α-LA is 45% helical and 9% beta sheet [10] and consists of two domains: a large alpha-helical domain and a small beta-sheet domain [9]. The large and small domains are connected by four disulfide bonds. The large domain includes three major alpha-helices from (residues 5-11, 23-24, and 86-98) and two short 310 helices (residues18-20, and 115-118) [9]. The small domain composes of a series of loops, a small three stranded anti-parallel beta-pleated sheets (residues 41-44, 47-50, and 55-56) and a short 310 helix (three residues per turn and an intrachain hydrogen bond loop with 10 atoms, residues 77-80) [9].
The two domains are stabilized and connected by four important disulfide bonds. The disulfide bonds between Cys-6 and Cys-120, Cys-28 and Cys-111 in the large domain and Cys-61-Cys-77 and Cys-73-Cys-91 in the small domain. The two disulfide bonds in the large domain are responsible for retaining about half the secondary and tertiary structure of the intact α-LA[13] while the other two disulfide bridges maintain the calcium-binding of the small domain. The small domain does not require the large domain to fold. However, its structure is significantly stabilized in the presence of the adjacent folded large domain [13].
α-LA can bind to several cations such as Ca2+, Mn2+, Mg2+, Na+ and K+. However, α-LA binds the strongest to calcium ions. Calcium binding is required for the formation of the correct disulfide bonds to re-generate the native structure from the reduced, denatured state. There are two binding sites for calcium. The primary binding site has a strong affinity for calcium ions. This binding site is formed by oxygen ligands from carboxylic groups of three Asp residues at 82, 87, and 88 and two carbonyl groups of the peptide backbone, Lys-79 and Asp-84, in a loop between two helices. Two water molecules coordinate the calcium ions [8]. Overall, the oxygen ligands form a distorted pentagonal bipyramidal structure. The apices of the pyramid are formed by carbonyl oxygen atoms while carboxylate groups and water molecules are arranged with the molecules approximately opposed. The side chain of the conserved Asp-83 points away from the calcium ion and is exposed to solvent [8]. The region in the vicinity of the calcium-binding loop is the most rigid part of Α-LA structure. The binding site of Ca comes from a loop formed on a tight turn similar to a beta-turn with an extra residue, Asp-82 acts as a “spacer” in the bend that links the two helices. This structure is similar to an “elbow” [8] [9].
The secondary calcium-binding site has a lower affinity for calcium ions and is found to be 7.9Å away from the primary calcium-binding site. The site is located near the surface of the α-LA molecule. Four residues are involved in Ca2+ coordination at this site in tetrahedral arrangement: Thr-38, Gln-39, Asp-83 and the carbonyl oxygen of Leu-81 [9]. No water molecules are present to coordinate the ion although there is one internal water 3.8 A from the secondary site [9]. The binding of the second calcium ion does not produce any significant structural change in α-LA.
There are also several zinc binding sites in α-LA. One of them is located in the “cleft” region of the protein. The strongest zinc-binding site is “sandwiched” between Glu-49 and Glu-116 of the symmetry-related subunit in the dimeric crystal unit cell [9]. Zinc- and calcium-binding sites can be occupied simultaneously. However, at room temperature and high zinc concentration, the binding of Zn+2 ions to Ca+2-loaded Α-LA decreases thermal stability, causes aggregation and increases its susceptibility to protease digestion [9].
Another important binding site is located 7.4Å away from the alpha-amino group [8]. The ligands for the Mn2+ ion are the side chain carboxyl groups of Lys-1, Glu-7 and Leu-11 and the peptide carbonyl group of Asp-84. However, the exact nature of the Mn+2 is not yet identified. A sulfate ion is also present in the Zn+2—α-LA structure at an intermolecular surface [8]. The ligands of the sulfate ion are two water molecules, Arg-70 from one molecule of α -LA and Gln-39 and Lys-94 from a symmetry-related molecule. The figure shown is meant to illustrate the relative locations of the residues on two α-LA molecules.
The functional site of α-LA includes part of the lower cleft of the protein with residues Phe-31, His-32, Leu-110, Gln-117 and Trp-118 being the key residues that maintain the structural stability of this site [7]. Mutagenesis of functional site residues indicates that Gln-117 and Trp-118 are specifically involved in the protein-protein interaction with GT while Phe-31, His-32 and Leu-110 influence the affinity of the LA—GT heterodimer for glucose [8].
The most important function of α-LA is its participation in the synthesis of lactose via its interaction with Galactosyltransferase. The mechanism of the interaction between GT and α-LA has yet been fully explained. According to the current hypothesis: α-LA and Galactosyltransferase form complexes with Mn2+ and UDP-galactose prior to the binding of monosaccharides [14]. When α-LA associates with GT, it reduces the reactivity of Lys-93 of one GT subunit and Lys-181 of another GT subunit while increases the reactivity of Lys-230, Lys-237, and Lys-241 which are in the catalytic domain of GT. These results α-LA plays a role in regulating the binding of the protein and its substrates.
It was hypothesized that the “cleft” region of the α-LA plays a major role in its regulatory function [2]. When bovine α-LA is tested, the α-amino group of Glu-1 in bovine α-LA (corresponds to Lys-1 in human) interacts with the side chain carboxyl oxygens of Asp-37 and Glu-39 (correspond to Asp-37 and Gln-39 in human α-LA). Since Asp-37 and Glu-39 are located at the entrance to the "cleft" region of α-LA, their interaction places Glu-1 in proximity to the association site of GT. The interaction between Glu-1 and a not yet known residue in GT induces a conformational change in the GT that increases its affinity for substrates [2]. The cleft region is an example of how α-LA's structure brings about its function. The cleft region of α-LA contains Tyr-103 which can interact with Trp-104 and Trp-60. The additional interaction between the two lobes restricts access to the upper cleft in α-LA. Therefore, the lower cleft in α-LA is partially blocked. The partially blocked cleft of α-LA is an example of how a protein's structure brings about its function. The blocked region allows the proteins to be more selective about which substrates can be bound to the protein. Only those that fit the blocked cleft can interact with α-LA. Surprisingly, the partially blocked cleft region is not a common feature even among proteins that show a high level of identity with α-LA.
For example, Type-C Lysozyme (1I22) in Homo Sapien has similar primary sequence to α-LA. Using PSI-BLAST to compare the two proteins, the E value is 5e-31[12]. PSI-BLAST is a program used to find proteins with similar primary sequence to a protein query. The E value is calculated by looking at the homology of the sequence and assigning gaps. The more homology sequence the α-LA has with the subjects, the lower the E value. The lower the E value, the more structural similarity the two proteins have. An E value below 0.05 is considered significant for those proteins [12]. In fact, α-LA and Lysozyme C are identical at 48 positions [4].
Except for the fact that Lysozyme C has a small alpha helix from residue 4 to 11 while alpha-Lactalbumin as a random coil around at these residues, the two proteins exhibit similar three dimensional structures. Both have three major alpha helices at about the same position and two anti-parallel beta-strands. The Z score for these proteins is 20.5 [5]. The Z score is obtained from the Dali Server. The Dali Server is a method for finding proteins with tertiary structure similar to the protein of interest. Dali measures the similarity between the two proteins by comparing the intramolecular distances. The measurements are given as Z scores. In contrast to the E score, the higher the Z scores, the more similarity the two proteins have. A Z score above two is considered significant.
Even though α-LA and Lysozyme C are similar structurally, they are highly divergent in function. α-LA regulates lactose synthesis while Lysozyme C (LZC) hydrolyzes the polysaccharide wall of bacterial cells [8]. Different forms of Lysozyme C are found in organisms other than mammals while α-LAs are only found in mammals, suggesting that α-LA arose through modifications of a duplicate of a gene for a calcium binding site in LZC [4].
α-LA differs from LZC in its stability and folding behavior. The native structure of α-LA is less stable than that of LZC and strongly depends on a tightly bound Ca+2 ion [7]. The main reason that explains the difference in structural stability between these two proteins is within the “cleft” region. The cleft region of LZC has Tyrosine at residue 103 instead of Proline like that of alpha-Lactalbumin. Originally, Tyr-103 in LZC does not interact with the residues of the β-lobe α-lobe as well [7]. As a result, no tight interactions occur and the cleft of LZC is available as the binding site for the extended oligosaccharide substrate instead of being partially blocked like in the case of α-LA. The sequence variation between these proteins reflects the relationship between functional and structural requirements during natural selection. The need for activity led to the availability for the entire cleft of LZC, while in α-LA, the need for native state stability lead to the blockage of the cleft by raising the degree of interactions between α-LA’s two domains.