Understanding the molecular dynamics!


Goals Accomplished by Modeling

Modeling is essential for understanding the interactions we cannot see with the naked eye. Our team found modeling extremely helpful in the times of COVID-19. Limited lab access made modeling our concepts more important than ever in order to understand what we wanted to produce in the lab. We used Pymol, a molecular visualization system, to understand the binding interactions between the carbohydrate binding module of interest and cellulose.

To gain a preliminary perspective on carbohydrate binding module (CBM) to cellulose binding, we modeled the interactions between the two using the software system Pymol. These models are useful in confirming that the CBMs will bind with cellulose since we have yet produced any viable CBMs for testing in the lab. Additionally, the models help visualize the nature of cellulose-CBM binding and what is occurring on the molecular level. The following models include our chosen CBMs, CBM2a and CBM3a, interacting with both bacterial cellulose (BC) and microcrystalline cellulose (MCC). Modeling CBMs using both BC and MCC is important as we used both forms of cellulose throughout our project.


Modeling Background

We chose CBM2a and CBM3a for testing since they are both categorized as Type A CBMs and have an extremely high affinity to crystalline cellulose. CBM2a can also bind irreversibly to amorphous cellulose but still traverse cellulose strands and cover a wider surface area without ever fully dissociating. Additionally all CBM2a’s share similar morphology: a β-barrel-type backbone with a relatively flat face on which there are several solvent-exposed aromatic amino acid residues that are involved in binding [1]. The CBM2a we have chosen was isolated from xylanase 10A of Cellulomonas fimi.

CBM3a was selected as we plan to test and improve the 2018 Toulouse iGEM team’s biobrick, Sirius. This CBM was isolated from Clostridium thermocellum which is thermostable. Thus, inclusion of CBM3a in our film might allow it to withstand higher temperatures. CBM3a’s surface has a planar region which is made up of polar and aromatic amino acid residues that have a high binding-affinity to cellulose strands [2].


CBM Electrostatics

Cellulose is generally negatively charged due to its abundance of oxygen atoms. This means positive areas of the CBMs bond with high affinity for these oxygens. As shown in our CBM3a electrostatics model, the planar binding face contains positive amino acid side chains, represented in blue, that bind to the negative oxygens on cellulose. Additionally, the hydrophobic tryptophan residues are suspected to interact with the apolar glucopyranoside ring of cellulose [1].

Figure 1: These models show the cellulose binding-surface of CBM3a. The spherical regions on the CBM3a indicate the amino acid residues that bind to the surface of cellulose. The main planar surface of the CBM3a contains polar amino acids which effectively anchor the CBM to cellulose strands and aromatic residues which are proposed to bind to crystalline cellulose. (A, B, and C) The same molecule viewed from different angles to better show the planar-binding surface of CBM3a.

Figure 2: CBM3a modeled with electrostatics to show the charges of the amino acid residues on its surface which bind crystalline cellulose. (A and B) CBM3a shown with surface electrostatics. Blue indicates amino acids with a positive charge and red represents amino acids with a negative charge. The white region contains neutral amino acids that do not have a high affinity for binding. (A) The planar binding surface of CBM3a where the blue regions represent the amino acids that will bind with a high affinity to negatively-charged oxygens of the hydroxyl groups on the cellulose monomers. (B) A 90° rotation of CBM3a to show the planar binding surface with polar amino acids represented as blue regions. (C and D) CBM3a is shown next to a bacterial cellulose crystal to model how CBM3a ought to interact with bacterial cellulose. The planar surface of CMB3a is shown facing the bacterial cellulose to model how it will bind to the cellulose. The electrostatics of cellulose are not shown and the red and green coloring of the crystal do not represent the charges of cellulose. (E) CBM3a is shown binding to a microcrystalline cellulose crystal to represent the difference in structure between microcrystalline cellulose and bacterial cellulose and how the CBM will bind. The planar binding face of the CBM is shown interacting with the cellulose strands with the polar amino acid residues having a high affinity towards the negative-regions of the cellulose.

Figure 3: CBM2a shown with its binding sites interacting with bacterial cellulose crystals. (A) CBM2a binding sites are given by the spherical regions on the model. These binding sites are composed of polar amino acids and aromatic residues that effectively bind to cellulose strands. These binding surfaces contain the amino acid tryptophan which contains aromatic rings that can bind cellulose.(B) CBM2a shown at a different angle to display the planar binding surface.(C) CBM2a is shown binding to a bacterial cellulose crystal while the red and green coloring of the crystal do not represent the charges of cellulose. (D) CBM2a is shown binding to the bacterial cellulose crystal structure from another angle. (E) CBM2a is shown with the electrostatics of its surfaces. The blue regions represent positive amino acid residues that are attracted to the negative oxygens from the hydroxyl groups on cellulose monomers. These two groups have a high-binding affinity for each other.