Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins

Figure 1. Organization of domains and motifs of Zyxin family proteins: the proline-rich region (PRR) and Lim1/2/3 domains. (NES = Nuclear export sequence, NLS = Nuclear localization Sequence, ZyM = Zyxin motif).

Figure 2. (A) The nuclear export sequences (NES) of Zyxin family proteins. To investigate the conservation of NES in proteins from other organisms, we aligned the human protein sequence using BLAST against the non-redundant protein sequence database. The resultant sequences obtained from BLAST were subjected to multiple sequence alignment, presented using WEBLOGO (B) The NES sequence for each protein was identified through the NetNES webserver, and the sequences were aligned using MultAlin. (C) WEBLOGO was used to investigate the conservation of residues in the sequences, which suggested the presence of signature LxxLxL/LxxxLxL, (L = L/V/I/F/M; x = any amino acid) repeat, in NES sequence across all the members of Zyxin family except zyxin and LPP which showed LxxxLxxL/LxxLxxL consensus sequence. (D) The nuclear localization sequence (NLS) for the Zyxin family members was fetched by the NLStradamus webserver with a prediction cut-off set at a recommended value of 0.6. The conservation of predicted sequences was analysed using MulitAlin.

Figure 3. Structures of Lim domains of the Zyxin family proteins. The structures were generated using comparative/homology modeling in Robetta server. Robetta server generates structure using structures identified by BLAST and PSI-BLAST using PDB database, the regions of the protein that could not be modeled using BLAST are modeled de-novo using Rosetta fragment insertion method. For each model, the N- and C-terminal are depicted in blue and red, respectively. All models were validated using PROCHECK and SWISS-MODEL workspace; and the structures with the highest Z-score are presented in Supplementary Figure S4.

Figure 4. Normal mode analysis (NMA) of Zyxin family Lim domain proteins. The NMA for each protein model was performed using the iMODS server under default settings. The motion of each domain is indicated by the arrows arising in the direction of motion. Additionally, the models are coloured based on NMA mobility of each structure, where the mobility is represented using a colour spectrum (blue represents the lowest mobility whereas red represents the highest mobility, and intermediate mobility is indicated by interim colours, blue>green>yellow>red). Interestingly, the direction of motion of all proteins is such that the distal ends of the protein move in the direction towards each other producing a motion that resembles clamping.

Figure 5. (A) Covariance analysis of the Zyxin family LIM domains. Covariance analysis was performed to understand the correlated movement of protein residues in the secondary structure which can shed light on the function of the protein. Each panel represents the covariance matrix for each member of the Zyxin family, in the matrix correlated motion is depicted as red, anti-correlated as blue, and uncorrelated as a white dot. High levels of correlated motions were observed in case of Lim1 domains of all protein proteins, varied amount of motion was observed in case of Lim2 with the highest in TRIP6, again Lim3 showed a similar kind of motion in all proteins. Proteins with similar localization and function demonstrated similarity in motion. For example, Ajuba, LIMD1, and WTIP displayed relatively similar covariance matrix of Lim3 domain these proteins are localized at the same site and constitute P-Body components, primarily perform gene regulation and mediate miRNA silencing. LPP and FBLIM1 showed similar correlated, uncorrelated and anticorrelated motions in Lim1/2/3, and both proteins involved mainly in cell shape maintenance; supporting the fact that similar dynamics may have similar…

Figure 6. Mutational frequency profiles of the Zyxin family proteins with different cancer malignancies. All proteins exhibit a considerable amount of mutational frequency which varied for each member. The highest mutation frequency was observed for Ajuba and the lowest was observed for LIMD1.

Figure 7. Pathway analysis by GeneMANIA. GeneMANIA highlighted the pathways that each protein member interacts within a cell. This helped us identify various proteins that interact with the query protein (A) Zyxin, (B) LimD1, (C) WTIP, (D) FBLIM, (E) AJUBA, (F) LPP and (G) TRIP6 . The connections are shown by blue lines, the width of each line represents the degree of interaction through weighted representation. The thicker line represents more interaction and vice versa.