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Function(s) and Biological Relevance

(Biofilm Associated Protein 1), which is a protein found in bacterial biofilms in Vibrio Cholerae, plays a significant role in the medical world. It is involved in the disease progression of Cholera. Bap1 is a lectin composed of two main structural units which include the β-prism domain and the β-propeller domain. It's main role is to bind citrate and carbohydrates, which occurs in the binding pocket of the β-prism domain. The article analyzed takes a further look into the structural function of the protein and its significance to overall biofilm adhesion.

Broader Implications

It is important to study Bap1 because of its clinical relevance with Cholera. 1.3-4.0 million people are diagnosed with cholera each year on a world wide scale, with it taking 21,000-143,000 lives each year.^[1] Cholera is transmitted indirectly through food and water that is contaminated with the Vibrio Cholerae bacteria. This is a huge issue considering that not everyone in the world has access to clean water. By understanding Bap1 and it's overall function, there is hope for figuring out how to break down it's bacterial biofilm and combat cholera.

Structural highlights and structure-function relationships

Secondary structure is important in Bap1. 6MLT is composed of two major tertiary structures which include the β-prism domain and the 8-bladed β-propeller domain. The β-prism domain is composed of twelve β-strands arranged into three antiparallel β-sheets with greek key folds.^[2] A greek key fold motif is a specific structural fold in a protein consisting of four adjacent antiparallel strands and their three linking loops.^[3]. The overall secondary structure is important in the β-prism domain in order to create a functional carbohydrate binding site. In the figure you can see the twelve β-strands in yellow, each with their perspective loops in white on the β-prism domain. The 8-bladed β-propeller also relies heavily on secondary structure for proper features. Each of the eight propeller blades consist of a four-stranded antiparallel β-sheet (yellow). ^[4] Bap1 is rich in β-sheets, which make up it's two main tertiary structures (β-prism domain and β-propeller domain). The function of the α-helix was not clearly identified. The model shows secondary structure with the β-helix in yellow, the α-helix in magenta, coils and loops in white, and turns in blue.

The β-prism and β-propeller are the two tertiary structures present in Bap1. These tertiary structures play significance roles in Vibrio Cholerae biofilm adhesion. The two domains are connected via two strands in between the five and six blade of the β-propeller, allowing for a great amount of flexibility between the two domains. The main function of the β-prism domain is to bind negatively charged citrate and sugar molecules. This contributes to the overall hydrophobicity of the biofilm, allowing for adhesive interactions with environmental surfaces. The β-propeller is composed of calcium/sodium binding motifs, which function by binding calcium and sodium ions at metal binding sites. The overall significance of ion binding to biofilm adhesion is not well known, but ions may play a role in structural stability of the β-propeller. The figure shows the β-prism domain and the β-propeller domain colored from the N (blue) to C (red) terminus. Note that Bap1 begins at the β-propeller (N 5') and continues on to the β-prism in between blades five and six and then returns and ends (C 3') at the β-propeller.

This image depicts the binding pocket in the β-prism domain. The structural makeup of the binding pocket carries relevance to the overall function of citrate and carbohydrate binding. The figure shows 6MLT in blue, with key amino acids highlighted in magenta, and a label of the specific binding site. It is helpful to use a of the binding pocket in order to see how the citrate and sugars would physically bind to the Bap1 protein.

A key characteristic of the binding pocket is its hydrophilic makeup, which plays a functional role in sugar binding. Hydrophilic amino acids in the binding pocket attract anionic sugars and citrate. The figure is shown in spacefill and is colored based on hydrophobicity. Hydrophilic residues are shown in blue, with hydrophobic residues in red, and non charged residues in white. Lys, which makes up a large part of the binding pocket is represented in lime green. The positively charged side chain on Lys makes it great for negatively charged sugars and citrate to bind to. If the binding pocket did not contain positively charged amino acids, sugar and citrate binding would not occur, resulting in a loss of function for Bap1.

The purpose of the blade motifs in the β-propeller is to bind calcium and sodium ions. Blade 1 coordinates two calcium ions via two intertwined calcium blade motifs, and the sodium ions are coordinated by individual calcium blade motifs in propeller blades 2-5 and 7.^[5] There is a possibility that the ions found in the β-propeller play a role in structural stability, rather than a functional or enzymatic role. ^[6] The figure shows where the metal binding sites occur in the β-propeller.

The catalytic triad has not been clearly identified. Asp 348, which is found on the beta-prism of Bap1, plays a crucial role in binding to citrate and carbohydrates. Mutation of aspartic acid to alanine results in a loss of function for Bap1. Since Ala has a much smaller side chain than Asp, it becomes too many Å away to interact with citrate and carbohydrates.

There are six key amino acids highlighted in the active site of 6MLT. They create interactions via hydrogen bonds or van der Waals in order to bind citrate and carbohydrates.

There are six important amino acids involved in the binding of citrate and sugars. These six amino acids include Gly 344, Ala 345, Val 346, Lys 501, Asp 348, and His 500. Gly 344, Ala 345, Val 346, Lys 501 all interact with citrate and sugars via hydrogen bonding, while Asp 348 and His 500 interact via van der Waals interactions. The amino acids are shown in CPK to highlight properties that correspond with hydrogen bonding.

Bap1 has shown to contribute to the hydrophobicity of Vibrio Cholerae biofilms^[7] by binding sugars and citrate. When Bap1 binds with anionic sugars, the polysaccharides coating the biofilm act as an adhesive to attach the colony to a surface. This prevents removal of the cells by physical force. It also prevents penetration of the biofilm by the immune system or antibiotics.^[8] If hydrophobicity of Bap1 became compromised, there could be a loss in biofilm adhesion function, which could be used to study treatments going forward for cholera. Pink represents the polar parts of Bap1, with gray showing the hydrophbic components of Bap1. The red sections represent water molecules.

Energy Transformation

Since the overall function of Bap1 is to hold together biofilms, there are no energy transformations present.