Figure 1.
Significant β-lactamase residues and its electrostatic characterization.
(a) Structure of a Class-A β-lactamase enzyme. The active site is located at the domain interface. The catalytic residue Ser-70 is shown in red. Other catalytic residues are shown in orange, whereas the Ω-loop is shown in blue at the top. Residues that maintain the structural integrity are shown in cyan. (b) Electrostatic potential values of ±1 kcal/mol are mapped to the protein surface. Red indicates negative potential, while blue indicates positive potential. The structure is oriented to display a patch of negative potential at the interface of the Ω-loop and helices H3, H4 and H6 that is conserved within the TEM/SHV enzymes. (c) Conserved electrostatic networks (cf. Figure 2b) are mapped to a BL structure. Green colored spheres represent α-carbons of residues interacting strongly with catalytic residues, which are highlighted in orange.
Figure 2.
Electrostatic properties of β-lactamase family.
(a) Multiple sequence alignment of twelve β-lactamases color-coded by shifts in residue pKa values from model values. Residues colored red express increased acidity, whereas residues colored blue show increased basicity. (b) Residues colored green exhibit strong (>|±1| kcal/mol) electrostatic interactions with catalytic residues that colored orange. The identified residues are also highlighted in the β-lactamase structure provided in Figure 1c. The Ω-loop region is indicated by the purple box. A cartoon representation of secondary structure is displayed on top of each alignment, while active sites are displayed below.
Table 1.
Summary of the active site electrostatic network1.
Figure 3.
Relationship between phylogeny and electrostatic potential maps.
The class-A β-lactamase family phylogeny is shown, which differentiates into 7 subgroups using a constant cut-level. Outgroups are represented by a unique color for better visualization. The structures closest to the phylogeny are oriented to highlight the active site region, which is indicated in green in the TEM-1 ortholog. Structures in the outer ring have been rotated in the y-direction by approximately 90 degrees, which highlights the Ω-loop region, also indicated in green. It is clear that structures from the same outgroup have visually similar electrostatic potential maps, whereas there are significant differences across the whole phylogeny.
Table 2.
Characterizations of charge and H-bond properties.
Table 3.
P-values from the statistical z-test comparing physiochemical patterns to two different clustering sets.
Figure 4.
Backbone flexibility of β-lactamases is well conserved.
(a) The flexibility for each structure is mapped onto the multiple sequence alignment of the class-A β-lactamase family. The backbone of residues colored red is flexible, whereas blue indicates rigidity. The spectrum bar illustrates the extent of flexibility and rigidity, which ranges from −1 to +1. (b) Flexibility index values averaged across the family are shown in in green, whereas the dashed lines highlight fluctuations (as defined by ±1 standard deviation). (c) Visual observation of backbone flexibility identifies three main flexibility regions that are mapped on to the structure. These flexible loops might have an important role in protein functionality.
Figure 5.
The backbone flexibility index reveals the nearly conserved isostatic nature of both (a) the Ω-loop and (b) the eight active site residues.
The black line indicates the average value. Marginal rigidity is able to maintain the active site structure, while also allowing for the flexibility needed for substrate recognition and catalysis.
Figure 6.
The phylogenetic tree along with the corresponding protein structures and cooperativity correlation plots.
Sequence and structure dynamics are evolutionary related as evident from cooperativity correlation clustering. Structures are color coded by backbone flexibility index, which illustrates that all β-lactamase family members are primarily rigid with some punctuating flexible loops. Conversely, pairwise allosteric couplings are overall varied, yet typically conserved within evolutionary outgroups.
Figure 7.
Conservation in H-bond networks.
(a) H-bond density is plotted per residue, which identifies regions rich in H-bond interactions. The Ω-loop is shown inside purple box and active site locations are marked as well. (b) Overlapped H-bond contact maps reveal three important sites important for maintaining the active site structure integrity and substrate catalysis. (c) The CA-CA atoms of residues at the corresponding sites (1, 2 and 3) are depicted by yellow, green and blue lines respectively. For better visualization only strong H-bond connections have been displayed on the structure.
Figure 8.
The best-fit heat capacity curve by mDCM is shown with usol = −2.61, vnat = −0.32 and δnat = 1.61, which are within normal ranges established by our previous studies (solid line = model and symbols = experiment).
The three fitting parameters are required to calculate free energy of the protein accurately using Eq. 1.
Figure 9.
All-to-all percent sequence identity (blue) and structural RMSD (red, in units of Å) are provided to highlight (dis)similarity.
Table 4.
Structural and catalytic characterization of the dataset.