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.
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 . 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 .
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 .
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.