Essential biochemical, biophysical and computational inputs on efficient functioning of Mycobacterium tuberculosis H37Rv FtsY

Int J Biol Macromol. 2021 Feb 28:171:59-73. doi: 10.1016/j.ijbiomac.2020.12.182. Epub 2021 Jan 5.

Abstract

Mycobacterium tuberculosis (M. tuberculosis H37Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include Vmax and Km for the hydrolysis of GTP by ftsY which were 20.25±5.16 μM/min/mg and 39.95±7.7 μM respectively. kcat and catalytic efficiency of the reaction were 0.012±0.003 s-1 and 0.00047±0.0001 μM/s-1 respectively. These values were affected upon changing the standard conditions. Cations (Mg2+ and Mn2+) play important role in FtsY enzymatic activity as increasing Mg2+ decrease the activity. Mn2+on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence.

Keywords: CD; FtsY; GTP; Mycobacterium tuberculosis; SRP pathway.

MeSH terms

  • Adenosine Triphosphate / chemistry*
  • Adenosine Triphosphate / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Biocatalysis
  • Cations, Divalent
  • Gene Expression
  • Guanosine Diphosphate / chemistry*
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / chemistry*
  • Guanosine Triphosphate / metabolism
  • Hydrolysis
  • Kinetics
  • Magnesium / chemistry
  • Magnesium / metabolism
  • Manganese / chemistry
  • Manganese / metabolism
  • Molecular Dynamics Simulation
  • Mutation
  • Mycobacterium tuberculosis / genetics*
  • Mycobacterium tuberculosis / metabolism
  • Protein Binding
  • Protein Biosynthesis
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / metabolism
  • Receptors, Cytoplasmic and Nuclear / chemistry*
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ribosomes / genetics
  • Ribosomes / metabolism
  • Signal Recognition Particle / chemistry*
  • Signal Recognition Particle / genetics
  • Signal Recognition Particle / metabolism
  • Signal Transduction
  • Substrate Specificity
  • Thermodynamics

Substances

  • 4.5S RNA
  • Bacterial Proteins
  • Cations, Divalent
  • FtsY protein, Bacteria
  • RNA, Bacterial
  • Receptors, Cytoplasmic and Nuclear
  • Recombinant Proteins
  • Signal Recognition Particle
  • Guanosine Diphosphate
  • Manganese
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • Magnesium