Biology subjects

Sankararamakrishnan, R.

Publications and source records attributed to Sankararamakrishnan, R..

6 recordsLinked to original sources

bioRxiv2026

Molecular mechanism of water and glycerol transport through hydrophobic selectivity filter in the aquaporin homolog of Trypanosoma brucei

The protozoan parasite Trypanosoma brucei is implicated in deadly African sleeping sickness. Experimental studies show that T. brucei codes for three aquaporin homologs (TbAQP1 to TbAQP3). TbAQP2 has been established as the high affinity drug transporter of drugs pentamidine and melarsoprol. Mutation in TbAQP2 or its loss result in pentamidine-melarsoprol cross-resistance. TbAQP2 is also shown to transport water, glycerol and other solutes to respond to osmoregulation in the infected hosts or glycerol metabolism. Experimentally determined structures of TbAQP2 shows that it adopts the same aquaporin-like hourglass helical fold. However, the so called aromatic/arginine selectivity filter (Ar/R SF) in TbAQP2 has neither arginine nor aromatic residue and all four residues are hydrophobic. Mutation and functional studies have demonstrated the role of Ar/R SF residues in the transport and selectivity of solutes in aquaporin homologs. The intriguing question is how the completely hydrophobic Ar/R SF region enables the transport of water and glycerol molecules. In this study, we used computational approach to elucidate the molecular mechanism of water and glycerol transport. Our equilibrium molecular dynamics simulations showed that the number of water molecules transported by TbAQP2 is almost one order of magnitude higher than that of prototype water channel AQP1. Moreover, the residence time within TbAQP2 channel is much less compared to that found in AQP1. The relatively wider constriction, interactions of water molecules with the selectivity filter residues and the contact duration, all contribute to a large number of water molecules transported through TbAQP2 channel. Our umbrella sampling studies show that when glycerol is transported through TbAQP2, it participates in interactions with channel residues that can be considered as complimentary to that observed in prototype glycerol transporter GlpF. Our studies reveal the molecular mechanism of water and glycerol transport in TbAQP2 and establish that TbAQP2 is an efficient water transporter. Statement of SignificanceTrypanosoma brucei causes African sleeping sickness and a homolog of aquaporin, TbAQP2, is involved in the transport of drugs that are used to treat this disease. Developing anti-parasitic drugs requires the knowledge of molecular mechanism of the proteins function. TbAQP2 has been shown to transport water and glycerol. Permeating solutes have to pass through a narrow constriction region formed by all hydrophobic residues. In the present study, equilibrium molecular dynamics simulations showed that TbAQP2 transports water molecules faster in large quantity in comparison with mammalian AQP1. Higher water transport is due to relatively wider constriction and minimum water interactions with selectivity filter hydrophobic residues. Permeating glycerol molecule is involved in complementary interactions with the channel residues. Our studies reveal how water and glycerol are transported through hydrophobic selectivity filter in TbAQP2.

biophysics↗

bioRxiv2023

Membrane-binding properties of NS1 proteins from Zika and Dengue viruses: Comparative simulations in explicit bilayers reveal significant differences

NS1 in flaviviruses is the only non-structural protein that is secretory and interacts with different cellular components. NS1 is localized in endoplasmic reticulum as a dimer to facilitate the viral replication. The crystal structures of NS1 homologs from zika (ZIKV) and dengue (DENV) viruses have revealed the organization of different domains in NS1 dimers. The {beta}-roll and the connector and intertwined loop regions of wing domains of NS1 have been shown to interact with the membranes. The membrane-binding properties and the differences between ZIKV and DENV NS1 homologs in interacting with the membranes have not been investigated. In this study, we have performed molecular dynamics (MD) simulations of ZIKV and DENV NS1 systems in apo and in POPE bilayers with different cholesterol concentrations (0, 20 and 40%). In the simulations with bilayers, the NS1 protein was placed just above the membrane surface. At the end of 600 ns production runs, ZIKV NS1 inserts deeper inside the membrane compared to the DENV counterpart. The conformational landscape sampled by NS1 in the presence of membrane was analyzed. Unlike ZIKV NS1, the orientation of DENV NS1 is asymmetric in which one of the chains in dimer interacts with the membrane while the other is exposed to the solvent. The {beta}-roll region in ZIKV NS1 penetrates beyond the headgroup region and some residues interact with the lipid acyl chains while the C-terminal region barely interacts with the headgroup. Specific residues in the intertwined region deeply penetrate inside the membrane with less interactions with water molecules. Our analysis showed that more charged residues of ZIKV NS1 are involved in stronger interactions with the headgroups than that found for DENV NS1. The role of hydrophobic and aromatic residues in interactions with acyl chain region is also evident. Presence of cholesterol affects the extent of insertion in the membrane and interaction of individual residues. This study clearly shows that the binding, insertion and interaction of ZIKV NS1 with the lipid bilayer significantly differs from its counterpart in DENV.

biophysics↗

bioRxiv2022

Dissociation of CED-4 from CED-9 upon EGL-1 binding: Molecular mechanism of linear apoptotic pathway in Caenorhabditis elegans

Many steps in programmed cell death are evolutionarily conserved across different species. The Caenorhabditis elegans proteins CED-9, CED-4 and EGL-1 involved in apoptosis are respectively homologous to anti-apoptotic Bcl-2 proteins, Apaf-1 and the "BH3-only" pro- apototic proteins in mammals. In the linear apoptotic pathway of C.elegans, EGL-1 binding to CED-9 leads to the release of CED-4 from CED-9/CED-4 complex. The molecular events leading to this process are not clearly elucidated. While the structures of CED-9 apo, CED- 9/EGL-1 and CED-9/CED-4 complexes are known, the CED-9/CED-4/EGL-1 ternary complex structure is not yet determined. In this work, we modeled this ternary complex and performed molecular dynamics simulations of six different systems involving CED-9. CED-9 displays differential dynamics depending upon whether it is bound to CED-4 and/or EGL-1. CED-4 exists as an asymmetric dimer (CED4a and CED4b) in CED-9/CED-4 complex. CED-4a exhibits higher conformational flexibility when simulated without CED-4b. Principal Component Analysis revealed that the direction of CED-4as winged-helix domain motion differs in the ternary complex. Upon EGL-1 binding, majority of non-covalent interactions involving CARD domain in the CED-4a-CED-9 interface have weakened and only half of the contacts found in the crystal structure between /{beta} domain of CED4a and CED-9 are found to be stable. Additional stable contacts in the ternary complex and differential dynamics indicate that winged-helix domain may play a role in CED-4as dissociation from CED-9. This study has provided a molecular level understanding of potential intermediate states that are likely to occur at the time of CED-4as release from CED-9.

biophysics↗

bioRxiv2021

Designing BH3-mimetic Peptide Inhibitors for the Viral Bcl-2 Homologs A179L and BHRF1: Importance of long-range electrostatic interactions

Viruses have evolved strategies to prevent apoptosis of infected cells at early stages of infection. The viral proteins (vBcl-2s) from specific viral genes adopt a helical fold that is structurally similar to that of mammalian anti-apoptotic Bcl-2 proteins and exhibit little sequence similarity. Hence vBcl-2 homologs are attractive targets to prevent viral infection. However, very few studies have focused on developing inhibitors for vBcl-2 homologs. In this study, we have considered two vBcl-2 homologs, A179L from African swine fever virus and BHRF1 from Epstein-Barr virus. We generated two sets of 8000 randomized BH3-like sequences from eight wild-type pro-apoptotic BH3 peptides. During this process, the four conserved hydrophobic residues and an Asp residue were retained at their respective positions and all other positions were substituted randomly without any bias. We constructed 8000 structures each for A179L and BHRF1 in complex with BH3-like sequences. Histograms of interaction energies calculated between the peptide and the protein resulted in negatively skewed distributions. The BH3-like peptides with high helical propensities selected from the negative tail of respective interaction energy distributions exhibited more favorable interactions with A179L and BHRF1 and they are rich in basic residues. Molecular dynamics studies and electrostatic potential maps further revealed that both acidic and basic residues favorably interact with A179L while only basic residues have the most favorable interactions with BHRF1. As in mammalian homologs, the role of long range interactions and non-hotspot residues have to be taken into account while designing specific BH3-mimetic inhibitors for vBcl-2 homologs.

biophysics↗

bioRxiv2019

Unraveling the substrate preference of an uncharacterized phylogenetic subgroup in Formate/Nitrite Transporter (FNT) family: Computational studies of anion transport in Escherichia coli FNT homolog

Formate/Nitrite Transporters (FNTs) selectively transport monovalent anions and are found in prokaryotes and lowers eukaryotes. They play significant role in bacterial growth and act against the defense mechanism of infected host. Since FNTs dont occur in higher animals, they are attractive drug targets for many bacterial diseases. Phylogenetic analysis revealed that they can be classified into eight subgroups and two of which belong to the uncharacterized YfdC- and YfdC-{beta} groups. Experimentally determined structures of FNTs belonging to different phylogenetic groups adopt the unique aquaporin-like hourglass helical fold. We considered formate channel from Vibrio Cholerae (VcFocA), hydrosulphide channel from Clostridium difficile (CdHSC) and the uncharacterized channel from Escherchia coli (EcYfdC) to investigate the mechanism of transport and selectivity. Using equilibrium molecular dynamics (MD) and umbrella sampling studies, we determined temporal channel radius profiles, permeation events and potential of mean force (PMF) profiles of different substrates with the conserved central histidine residue in protonated or neutral form. Unlike the VcFocA and CdHSC, MD studies showed that the formate substrate was unable to enter the vestibule region of EcYfdC. Absence of a conserved basic residue and presence of acidic residues in the vestibule regions, conserved only in YfdC-, were found to be responsible for high energy barriers for the anions to enter EcYfdC. PMF profiles generated for ammonia and ammonium ion revealed that EcYfdC can transport neutral solutes and could possibly be involved in the transport of cations analogous to the mechanism proposed for ammonium transporters. Although YfdC members belong to the FNT family, our studies convincingly reveal that EcYfdC is not an anion channel. Absence/presence of specific charged residues at particular positions makes EcYfdC selective for neutral or possibly cationic substrates. This adds to the repertoire of membrane proteins that use the same fold but transport substrates with different chemical nature.\n\nAuthor SummaryChannels and transporters are membrane proteins involved in the transport of solutes selectively across the cell membranes. Drugs for many diseases have been developed to inhibit ion channels. Formate/Nitrite Transporters (FNTs) are ion channels selective for monovalent anions and are present in bacteria and lower eukaryotes. Absence of FNTs in humans makes them as attractive drug targets against many pathogenic bacteria. To develop inhibitors for a protein, it is important to understand the mechanism of its function. Selectivity and transport mechanism of FNTs have been investigated for some members. One of the subgroups of FNTs, YfdC-, is uncharacterized. In this study we used computer simulation approach to investigate the molecular mechanism of selectivity and transport of three FNTs including one from YfdC- group from Escherichia coli. Our studies show that E. coli YfdC is not an anion channel although it belongs to FNT family. We hypothesize that the YfdC- members could be involved in the transport of neutral or possibly cationic substrates. This is further supported by the conservation of specific acidic residues found only in YfdC- in the vestibule regions. This finding has major implications in developing blockers for FNT members belonging to YfdC- group.

biophysics↗

bioRxiv2017

Cooperativity in Plant Plasma Membrane Intrinsic Proteins (PIPs): Mechanism of Increased Water Transport in Maize PIP1 Channels in Hetero-tetramers

Plant aquaporins (AQPs) play vital roles in several physiological processes. Plasma membrane intrinsic proteins (PIPs) belong to the subfamily of plant AQPs and they are further divided into two closely related subgroups PIP1s and PIP2s. Members of the two subgroups have been shown to have different transport properties. While PIP2 members are efficient water channels, PIP1s from some plant species have been shown to be functionally inactive. Aquaporins form tetramers under physiological conditions. PIP2s can enhance the water transport of PIP1s when they form hetero-tetramers. However, the role of monomer-monomer interface and the significance of specific residues in enhancing the water permeation of PIP1s have not been investigated at atomic level. We have performed all-atom molecular dynamics (MD) simulations of homo-tetramers of ZmPIP1;2 and ZmPIP2;5 from Zea mays and hetero-tetramers containing ZmPIP1;2 and ZmPIP2;5 with different stoichiometries and configurations. ZmPIP1;2 in a tetramer assembly will have two interfaces, one formed by transmembrane segments TM4 and TM5 and the other formed by TM1 and TM2. We have analyzed channel radius profiles, water transport and potential of mean force profiles of ZmPIP1;2 monomers with different types of interfaces. Results of MD simulations clearly revealed that TM4-TM5 interface and not the TM1-TM2 interface is important in modulating the water transport of ZmPIP1;2. We generated in silico mutants of specific residues that are involved in contacts with adjacent monomers. MD simulations of mutant tetramers highlighted the importance of I93 residue from the TM2 segment of ZmPIP2;5 for the increased water transport in ZmPIP1;2.

biophysics↗