IN SILICO FUNCTIONAL ASSIGNMENT TO Rv3430c, A Mycobacterium tuberculosis

GENE OVEREXPRESSED DURING INFECTION IN BLOOD FROM HIV+/- SUBJECTS

Panjab UniversityChandigarh

Presented by:Rajni Devi

M.Sc. Biochemistry

A transcriptomics experiment conducted by Ryndak et al, in 2014 on M. tuberculosis

genes in HIV+ and HIV- patients was retrieved.

A set of genes that were significantly overexpressed during pathogenesis

(HIV+/HIV-) were analysed.

There were different ‘Rv’ genes with no known function which showed significant

increase in expression upon HIV+/HIV- patients.

Rv3430c was selected for analysis. It showed a 5-fold and 3-fold increase in

expression in HIV+ and HIV- infection respectively.

Introduction

Objectives

Amino acid sequence based functional assignment.

Build an 3D computational model of Rv3430c.

Comparison of Rv3430c model with other known proteins structures.

Identification of active site residues in Rv3430c.

Analysis of the binding pocket.

Screening of genes

(UniProt)

Rv3430c Sequence

based analysis (Pfam, SCOP)

Structure Visualization and analysis

(ICM)

Structure Validation (VADAR)

Search for homologs (BLAST)

3-D structure prediction

(LOMETS)

Structure Super-

positioning (Dali Server)

Active site & binding pocket

analysis (ICM

Browser)

Methodology

RESULTS

Rv3430c Protein Sequence

Rv3430c is 387 amino acid sequence. This amino acid sequence was used for further analysis to predict the function of Rv3430c gene

Structural and Evolutionary relationship amongst known protein

SCOP predicted Rv3430c belonged to a Superfamily called ‘Ribonuclease H-Like’ which have Ribonuclease H- like domain.

Visualization of Rv3430c protein structure

N-terminal domain (right side) have a HHCC motif. Catalytic core domain (central) is integrase domain and it contain a DDE motif. C-terminal (left side) non specifically

bind to DNA and contain SH3 motif.

Search for structure homologs

Dali Server generated a large number of structures with coordinates transformed according to the submitted model. Out of these 8 structures were selected for

superposition based on lower RMSD.

Structures RMSD in (A )⁰ Seq. Identity (%) Name Of The Organism

4e7h_A (Red) 1.4 14 Human spumaretrovirus

4eb2_A (Blue) 1.4 14 Human spumaretrovirus3oS0_A (white) 1.3 15 Simian foamy virus

1cxu_A (Orange) 2.1 21 Avian sarcoma virus

1asv_A(Cyan) 2.3 20 Avian Sarcoma Virus IN

n1c0m_A (Pink) 2.4 20 Rous sarcoma virus1asu_A (Green) 2.6 19 Avian sarcoma virus

1biu_A (Maroon ) 2.8 19 Human immunodeficiency virus 1

Structure Superposition

Rv3430c (yellow) The C-terminal region important for DNA binding and catalysis superposed well with

other integrases, inspite of no significant sequence identity.

Structure based Sequence Alignment

Arrow and cylinders depict β-strands and α-helix respectively. The DDE region (shaded cyan) are conserved in all integrases and active site residues ( shaded yellow).

Active site amino acid residues analysis

The active site residues were Asp145, Asp208, Ser232, Tyr234, His235, Asn240, and Glu244. These residues were conserve in all other integrases. This predicted that

Rv3430c is probably an ‘Integrase’

Manual Docking with DNA

Binding pocket for DNA in A: 3oS0_A, B: 4e7h_A, C: 4be2_A and D: Rv3430c. All the structures have conserved active site, among these in spite of low sequence identity.

Summary

• This work was aimed at assigning function to an unknown M. tuberculosis gene

whose expression goes up during HIV+/- subjects.

• Rv3430c was subjected to various bioinformatic analysis.

• Pfam predicted gene to be an Integrase.

• The structural analysis showed that Rv3430c has a typical conserved DDE region,

common to other integrases.

• Based on the active site analysis and comparison with known structures, it was found

that in spite of no significant sequence identity, the active site residues are conserved.

• This study shows that Rv3430c is likely to be an Integrase.