• Molecular dynamics simulations (Familiar with GROMACS and PLUMED software packages).
• Molecular docking (Familiar with Autodock, Autodockvina software).
• Visulalization and analyses of biomolecules (Familiar with VMD and Chimera software).
• Modelling proteins and loop regions of proteins using Modeller.
• Preliminary knowledge on QM/MM simulations. (Exploring CP2K-GROMACS interface ).
• Ready to embrace any new field.

• Upendra, N., Abhishek, A., Balaji, P., Krishnaveni, S. Conformational Studies on Bacillus subtilis RbgA using Molecular Dynamics Simulations. International conference on Recent Advances in Materials Science and Biophysics, January 23-25, 2018, Mangalore University, Mangaluru.
• Upendra, N., Abhishek, A., Balaji, P., Krishnaveni, S. Molecular Dynamics Simulations on EngA – A GTPase involved in Ribosome Assembly. International Conference on Advanced Functional Materials for Energy, Environment and Health Care (AFMEEHC), March 18-20, 2019, Vijnana Bhavan, University of Mysore, Mysuru.
• Upendra, N., Abhishek, A., Balaji, P., Krishnaveni, S. Conformational Studies on EngA GTPase using Molecular Dynamics Simulations. National Seminar on Biomolecular structure and dynamics, March 28-29, 2019, Periyar University, Salem.
• Upendra, N., Krishnaveni, S. Molecular Dynamics Simulations on Bacillus subtilis EngA – For Exploring Nucleotide Dependent Conformations. Conference on Drug Design and Discovery Technologies, November 21-22, 2019, Ramaiah University of Applied Sciences, Bengaluru.
• Kavya K. M., Upendra N., Krishnaveni S. Surface exposure studies on Thermotoga maritima YsxC in GDP and GTP-Mg2+ bound states using molecular dynamics simulation. International Symposium on Emerging Materials for Sustainable Energy and Environment (EMSEE-2023) July 15, 2023.
• Kavya K. M., Upendra N., Krishnaveni S. Conformational studies on Bacillus subtilis YsxC in GDP and GTP-Mg2+ bound states using molecular dynamics simulation. International Conference on Modern Functional Materials and Its Multifunctional Applications (ICMFM-2023) July 21st – 22th, 2023.
• Kavya K. M., Upendra N., Shuchika D. Biligere., Krishnaveni S.Investigation of Phe-tRNA interaction with EF-Tu in GDP/GTP Nucleotide bound states: A molecular dynamics simulation study. Indian Conference on Bioinformatics 2023 (Inbix’23) November 24th – 26th, 2023.

During My Ph.D. period, I have carried out molecular dynamics (MD) simulation studies on RA- GTPases (ribosome assembly GTPases). RA-GTPases are GTP binding proteins that are involved in the different stages of ribosome biogenesis. How exactly these GTPases aid in ribosome biogenesis at the molecular level helps us to regulate cell proliferation (that leads to the colonization of harmful bacteria or the growth of cancerous cells) through the regulation of ribosome biogenesis. Since the ribosome is a protein factory that synthesizes proteins, they are the essential ingredients for cell growth, multiplication, and sustenance.

Basic structural features of GTPases and their associated functions and questions to be addressed.

GTPases are also called as GTP binding proteins or G-proteins which constitute an important class of regulatory proteins that bind and hydrolyses GTP into GDP (guanosine-5′- diphosphate) and inorganic phosphate. GTPases bind guanine nucleotides (GTP/GDP) within the GTP- binding domain or G-domain. This is having an α/β fold which typically has a central β-sheet of at least 6 (mostly parallel) β-strands surrounded by 5 – 6 α-helices. G-domains possess 5 sets of a sequence of amino acids which is conserved in all GTPases called G-motifs: G1 [GxxxxGK(S/T)], G2 [T], G3 [DxxG], G4 [(N/T)(K/Q)xD] and a weakly conserved G5 [SAK] motif. These G-motifs facilitate interactions with the bound guanine nucleotide. The G4 and G5 motifs interact and stabilize the guanine base. The G1 motif facilitates interaction with the phosphate moieties, thus the loop of the G1 motif is referred to as P-loop (phosphate-binding loop). The G2 and G3 motifs tend to interact with gamma- phosphate. As a consequence, the loops of G2 and G3 motifs may undergo large conformational changes when GTP is hydrolyzed into GDP. This in turn induces conformational changes in effectors of GTPases (neighboring molecule: protein or RNA ) and activates its function. Therefore, the loops of G2 and G3 are termed switch-I (Sw-I) and switch-II (Sw-II) respectively. The variation in conformational changes at switch loops may vary among different G-proteins, some exhibit larger fluctuations, while others show only small fluctuations. These variations probably depend on the length of the loops and their positions in the environment (bio-molecular complex). Some G-proteins show an effect on a distantly positioned site from GTP binding site upon switching from GTP-bound (on/active) to GDP-bound state (off/inactive). Therefore, GTPases are molecular switches that regulates many biological functions such as cell multiplication, signal transduction, protein synthesis and ribosome biogenesis, etc., through switching between the on and off states.

GTPases involved in ribosome biogenesis are referred to as RA-GTPases. RA-GTPases such as RbgA, ObgE, and YsxC are involved in the maturation of the 50S ribosomal subunit whereas RsgA, Era, and YqeH are involved in the maturation of the 30S ribosomal subunit. EngA, YihA, and Obg are involved in the maturation of both the 50S and 30S subunits. Depletion of any of these factors results in the incomplete formation of the ribosome. Therefore, binding of these GTPases to the intermediate ribosomal subunits is essential to mature into a complete functional ribosome. However, it is reported that binding of these GTPases to the ribosomal intermediate depends on its bound-nucleotide (GTP/GDP). This nucleotide dependent association may be attributed to the conformational shifts that occur between the GTP-bound state and the GDP-bound state of the GTPase. In line with this, many structural studies have been conducted and obtained structures of RA-GTPases bound to different nucleotides. Among these, few GTPases show no significant conformational differences between different nucleotide bound states in contrast to their counter experimental suggestions. Some GTPases exhibit large conformational rearrangement between the different species of the of the same GTPase with distinct bound states. For example, the GTPase EngA, which contains two G-domains, exhibits different conformational arrangements for different species with distinct nucleotide bound states. Whether this conformational change is due to different species or different nucleotide bound states is yet to be answered. In addition, the comparison of Bacillus subtilis EngA structures of different nucleotide bound combinations show no significant conformational variations but exhibited different conformations from SAXS (small angle X-ray scattering) profiles. Similarly, another GTPase, RbgA from Staphylococcus aureus show no significant conformational differences between different nucleotide bound states but exhibited different patterns in HDX-MS spectroscopy.

The above facts and gaps on RA-GTPases prompted me to explore the conformational variations of RA-GTPases in different nucleotide bound states and also instigated to explore the interactions of the RA-GTPases with the (i) ribosome, (ii) ribosome constituents (rproteins/rRNAs), and (iii) other RA- GTPases or other ribosome maturation factors.

As a starting point, I explored the conformational variations of EngA and RbgA in different nucleotide bound states and mutations for different species. These studies are compiled in my Ph.D. thesis.

However the interaction of RA-GTPases with the (i) ribosome, (ii) ribosome constituents (rproteins/rRNAs), and (iii)other RA-GTPases or other ribosome maturation factors need to be explored in furure.

Summary of the MD simulation studies on EngA

EngA, a GTPase contains two consecutive G-domains [GD1, GD2] at the N-terminal and C- terminal K-homology (KH) domain postulated to bind with an rRNA. The nucleotide bound combinations of EngA dictates its association with the ribosomal subunits. (i) [apo, GTP] and [GTP, GTP] bound combinations bind to the 50S subunit, (ii) [GDP, GTP] bound state binds to the 30S subunit and (iii) when the second G-domain is devoid of GTP, EngA will not bind to any of the subunits. This nucleotide dependent EngA-ribosome association is attributed to the conformational changes between different nucleotide bound states.

This motivated to explore conformational variations of EngA in different nucleotide bound combinations using all-atom MD simulations.

MD simulations on Thermotoga maritima EngA (TmDer) for 500 ns in different nucleotide bound states exhibit that the nucleotide bound state of the second G-domain (GD2) influences the GD1-KH interface interactions. This indicates an allosteric connection between the GD2 pocket and the GD1-KH interface regions. This connection is further observed through in silico mutation studies. (This work is published: Upendra, N., Krishnaveni, S. Molecular dynamics simulation study on Thermotoga maritima EngA: GTP/GDP bound state of the second G-domain influences the domain- domain interface interactions. Journal of Biomolecular Structure and Dynamics 5, 1–13 (2020). DOI: 10.1080/07391102.2020.1826359.Impact factor 5.235 )

Next, the conformational variations of Bacillus subtilis EngA (YphC) in different nucleotide bound states along with the presence or absence of Mg2+ at the active-site were explored using 1000 ns MD simulations. This study emphasizes that the presence of Mg2+ organizes the geometry of the active-sites of G-domains such that the probability of GTP hydrolysis is more favorable in GD1 domain as compared to GD2 domain. Interestingly this is in accordance with the already reported experiment. (This work is published: Upendra, N., Kavya K.M., Krishnaveni, S. Molecular dynamics simulation study on Bacillus subtilis EngA: The presence of Mg2+ at the active-sites promotes the functionally important conformation. Journal of Biomolecular Structure and Dynamics. DOI: 10.1080/07391102.2022.2151513. Impact factor 5.235)

Summary of the MD simulation studies on RbgA

RbgA, a GTPase contains an N-terminal G-domain and the C-terminal ANTAR (AmiR and NasR transcription antitermination regulators) domain. RbgA is shown to be involved in the late stages of the 50S ribosomal subunit maturation. RbgA-ribosome association is more in GTP bound state and very weak or no association in GDP bound state of RbgA. This is attributed to the conformational changes between the nucleotide bound states.

This motivated to explore the conformational dynamics of RbgA in various nucleotide bound states along with the presence or absence of Mg2+ using all-atom MD simulations.

Initially, MD simulations of Staphylococcus aureus RbgA (SaRbgA) in various nucleotide bound states along with the presence or absence of Mg2+ at the active-site were performed for 1000 ns. This study revealed that the nucleotide bound state dictates the secondary structural arrangement of the C- terminal tail and hence its mobility. This indicates that the C-tail of SaRbgA probably interacts with rRNA, which is also realized through in silico mutation studies. The connection between the GTP binding site and the C-tail were investigated through protein energy network analysis for the representative conformations of the different nucleotide bound states. The representative conformations for different nucleotide bound states were extracted through the free energy landscape method. In addition, the presence of Mg2+ at the active-site influences the Sw-I mobility and stabilize the active-site water molecules which probably aids in GTP hydrolysis.

(This work is published: Upendra, N., Krishnaveni, S. Conformational exploration of RbgA using molecular dynamics: Possible implications in ribosome maturation and GTPase activity in different nucleotide bound states. Journal of Molecular Graphics and Modelling 111, 108087 (2022). DOI: 10.1016/j.jmgm.2021.108087. Impact factor 2.942)

Next, MD simulations of Bacillus subtilis RbgA (BsRbgA) in different nucleotide bound states along with the presence or absence of Mg2+ at the acitve-site were carried out. Network analyses of the simulations reveals that different nucleotide bound states influences distinct sites. Further, the positive impact of Mg2+ on GTP hydrolysis has been observed. (manuscript is under preparation)

In addition I have also carried out MD simulations on single stranded G-qudruplex DNAs after docking with flouscent probes. This work is published in colloboration with the experimental team (DOI: 10.1016/j.bmc.2021.116077).

Future perspectives

I intend to explore the interaction of RA-GTPases with the (i) ribosome, (ii) ribosome constituents (rproteins/rRNAs), and (iii) with other RA-GTPases or other ribosome maturation factors in order to delineate the ribosome maturation process at the molecular level. This can be conducted through systematic study of protein-protein and protein-RNA docking, which is then followed by coarse-grained simulations.

Long term goal: I would like to involve and develop the multi-scale modeling method which integrates different dimensional mechanisms such as quantum mechanics, all-atom and coarse grained molecular mechanics, and continuum mechanics to address time to time societal requirements.