THE STORY AND POTENTIAL OF ALPHA-LACTALBUMIN
This Jmol Exploration was created using the Jmol Exploration Webpage Creator from the MSOE Center for BioMolecular Modeling.
Alpha-lactalbumin is a calcium binding, low molecular weight metalloprotein that is composed of 124 amino acids.
View LALBA!This protein has two secondary structure domains: an alpha domain (red) and a beta domain (blue) divided by a deep cleft.
View Alpha & Beta Domains!The alpha domain (red) is very large, and is composed of 5 alpha helices.
LALBA has three major alpha helices (hot pink) ...
...And three short 3/10 helices (light pink).
View Minor Alpha Helices!The h1a 3/10 helices 13-15,(light pink)lies the hydrophobic core 13-18 (medium violet red). Tryptophan 26 in human alpha-lactalbumin is replaced by a leucine, resulting in a slight rearrangement of the alpha domains hydrophobic core compared to other mammals. This change in side chain is compensated by methionine in residue 30 in humans.
View h1a 3/10 helix, hydrophobic core, & Trp26Question: What do you think will be the impact of the switch from tryptophan to leucine in human LALBA?
Answer: Tryptophan has a bulky hydrophoibic side chain, while the non-polar side chain of leucine is very small in comparison, which results in less interactions within the hydrophobic core, leading to a less tightly packed hydrophobic core.
Question: What type of molecules do you think could bind to the hydrophobic core?
Answer: Carbohydrates non-polar rings and fatty acids hydrocarbons.
The ACI is located in the alpha domain of LALBA. The ability of LALBA to regulate the substrate specificity of glycotransferase (GT) in lactose synthesis is associated with the ACI and the flexible loop regions. The ACI is composed of invariant residues Phe31, His32, Gln117, and Trp118.
Phe31 and His 32 are located at the end of the helix H2 immediately adjacent to the lower regions of the cleft, and Gln117 and Trp118 are located in the C-terminal tail of LA.
Phe31 and Gln117 are relatively exposed, whereas His32 and Trp118 are more shielded from solvent. The location of His32 is influenced by the conformation of the flexible loop region which will be discussed later. This region is flanked by two amino groups (Lys5, and Lys 114).
View ACI Sided by Lys5 and Lys114!Question: There are interactions that can be observed between Gln117 and Lys5, as well as Gln117 and Lys114. What type of intermolecular interactions are present, as well as what type of structure is the result of these interactions (1, 2, or 3)?
Answer: The interactions that can be observed between Gln117 and Lys5, as well as Gln117 and Lys114 are hydrogen bonding forces. These forces impact the tertiary structure of the molecule because of the side chain-backbone interactions that can be observed: backbone of Gln117 and side chain of Lys5, as well as side chain of Gln117 and backbone of Lys114.
Another region within the alpha domain of LALBA that is essential to the function of alpha-lactalbumin is the flexible loop. This region is adjacent to the lower end of the cleft, it sides the ACI, and it interacts with the C-terminal end of the helix H2.
In humans, the polypeptide backbone of this region adopts a distorted alpha-helical conformation (H4b). This is region is known to be structurally heterogeneous in other mammals due to varied pH conditions, however, chemically, (aside from residue 109), residues 104-111 are known to be conserved in all mammalian species. In humans, the pH impacts residue HIS107 because it interacts via hydrogen bonding with the carboxylate group of Glu25.
The flexible loop is known to have varied conformation depending on solution it is in. It has been observed in both helical and loop crystal forms in humans at pH 6.5 at room temperature. The conformation impacts the tertiary structure of the region as well because of how the residues will interaction.
Question: What do you think would happen to the interaction between His107 and Glu25 at low pH?
Answer: At low pH, ions may be protonated due to acid conditions, so this could interfere with hydrogen bonding possibility between the histidine and glutamic acid carboxylic acid that is protonated. This will result in a helix turning into a coil to locate the protonated histidine in the acidic solvent environment.
The beta domain is very small, and is composed of 3 beta sheets. It has little regular secondary structure because it is composed of a series of loops with small, three stranded, antiparallel pleated sheets (S1 41-44, S2 47-50, and S3 55-56)...
View Anti-parallel Pleated Sheets!...And a short 3/10 helix (h2-77-80).
View h2 3/10 helix!Question: What is the advantage of antiparallel pleated sheets vs. parallel pleated sheets for secondary structure?
Answer: Antiparallel pleated sheets are more stable than parallel because of direct interactions between the carbonyl and amide backbones of amino acids, whereas parallel pleated sheets have more difficulty forming hydrogen bonding because of increased distance between the backbones of polypeptide chains.
It is important to note that H1-3, S1-3, h1b, h2, and h3c are essential for LA function and are conserved within all mammals.
In mammary glands of mammals, LALBA is unable to bind to monosaccharide in isolation, however, when it forms and activates the lactase synthase enzyme with a glycol-transferase, the cleft region is known to stabilize monosaccharide binding. When bound with GT, LALBA increases the affinity for glucose to bind.
In nature mono-saccharide binding sites are found to be between two domains of a protein and is mediated by hydrogen bonding between the simple sugars hydroxyl groups and polar groups of the protein, as well as by non-polar interactions between aromatic side chains of the residues in the binding site and the sugar rings of the simple sugar.
The following residues in LALBA,(that can be found in sub-site F of C-type lysosomes), are involved in monosaccharide binding: Phe31, His32, and Leu110.
Question: Why do you think that residues Phe31,His32, and Leu110 are important in monosaccharide binding?
Answer: Monosaccharide binding is mediated by hydrogen bonding between sugar hydroxyl groups and polar groups in the protein, as well as by non-polar interactions between aromatic side chains of protein residues and the sugar rings.
Histidine is slightly basic, Phenylalanine has a large non-polar side chain, and Leucine also has non-polar regions.
Like interacts with like, and carbohydrates have many non-polar hydrocarbon regions that could interact with the phenylalanine and leucine side chains.
Histidine can also interact through hydrogen bonding interactions with a saccharide because of its basic side chain.
The cleft is a known region in C-type lysosomes, which is comparable to the cleft region of LALBA. In LALBA, the cleft is shortened due to the blockage of two (A and B) of the six sub-sites (A-F) by tyrosine103.
View Tyrosine103 Blocking!Sub-site F is located at the lower end of the cleft region where it broadens out the proteins surface and is relatively shallow and accessible to solvent unlike C and D sub-sites. The shape of F is also influenced by the conformation of the flexible loop.
Question: Why is the shape of sub-site F of the cleft region influenced by the conformation of the flexible loop?
Answer: Leu110 is part of the flexible loop as well as a component of the subsite F.
The F sub-site is more accessible to solvent when the flexible loop residues 105-110 have a helical conformation in humans than a loop conformation in other mammals. However, the functional significance of the flexible loop shape to monosaccharide binding is unknown.
LALBA has a low affinity (low likelihood to bind) for other metal ions to bind in the calcium binding site.
The calcium binding site stabilizes the protein against denaturation at different temperatures. The calcium ion interacts with the helices found between the alpha and beta domains, and this calcium ion is held by seven oxygen groups.
- Two of these oxygen are found in the backbones of Lys78 and Asp 84.
-Three oxygen are located at the side chains Asp 82, 87, &88.
-The last two oxygen that hold the calcium ion are supplied by two water molecules.
Question: Why does the calcium ion help stabilize the protein against denaturation?
Answer: The calcium ion interacts with seven oxygen around it through ionic interactions. These interactions are more difficult to disrupt, thus requiring more energy to be broken, helping to protect the protein against denaturation at high temperature.
Also, the location of the calcium ion is between two helices, which are located between the alpha and beta domains. The calcium ion holds the helices close to it through the ionic interactions. The helices interactions thus become closer together, compressing the protein through closer interparticle forces that will be harder to disrupt.
This protein has a low affinity (low likelihood to bind) for other metal ions to bind in the calcium binding site.
Question: Why do you think that is?
Answer: LALBA has a higher affinity for calcium ions. This is due to the atomic radius of calcium being large enough for the calcium ion to bind with the seven oxygens.
For example, if Mg2+ were to try to replace a calcium ion, it would not be able to interact with all seven oxygens due to its smaller atomic radius than that of calcium, making it more difficult for the magnesium ion to bind correctly and hold the protein together.
Also, the other metal ions, such as Mg ion, cannot displace calcium to bind with LALBA (when the calcium ion is already bound to the protein). Furthermore, even if the calcium binding site does not have calcium ion present, the other metal ions are unable to alter the native conformation of the binding site.
Question: Would the calcium binding site be considered a primary, secondary, or tertiary structure?
Answer: The Calcium binding site is a tertiary structure because it is a prosthetic group of LALBA. The backbone interactions this group uses are also considered tertiary structure because this group is not a part of the protein to use secondary interactions with the protein.
LALBA is a known protein found in breast milk. Studies have shown that a complex of oleic acid bound to alpha-lactalbumin has resulted in significant treatment of persistent warts. The LALBA-oleic acid protein-lipid complex is utilized when traditional treatments fail for stubborn warts.
Further studies in mice suggest possible use in cancer therapies because of the LALBA-oleic complex known abilities to activate apoptosis of tumor cells.
Gustafsson, L. (2004). Protein in breast milk might treat warts. New England Journal of Medicine. 350(2603-2672). Retrieved from http://www.m.webmd.com/skin-problems-and-treatments/news/20040623/protein-in-breast-milk-might-treat-warts?page=1
Pike, C. W., Brew, K., & Acharya, K. R. (1996). Crystal structures of guinea-pig, goat and bovine alpha-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase. Structure, 4(6), 691-703. doi: 10.2210/pdb1hfz/pdb