Indira H. Shrivastava, PhD

Department of Computational & Systems Biology

School of Medicine

Pittsburgh PA 15213

voice: 412 383 5806

fax: 412 648 3163


Research Collaborations

Prof. Susan G Amara (Glutamate Transporters)

Prof. Ingo Greger ( Ionotropic Glutamate Receptors)

Judith M Lalonde (HIV Glycoprotein GP120)

Prof. Patrick Thibodeau (RTX-toxins)

Prof. Glorioso (HSV Glycoproteins)



Molecular Modelling and Simulations of Membrane Proteins

Molecular dynamics simulations are widely used to obtain information of the time evolution of conformations of proteins and other

biological macromolecules. Simulation trajectories provide atomic level detail concerning the motions of individual particles

as a function of time. They can be utilized to quantify the properties of a system at a precision and on a time scale that is otherwise

inaccessible, and is therefore a valuable tool in extending our understanding of model systems. Molecular dynamics simulations, along

with a range of complementary computational approaches, have become valuable tools for investigating the basis of protein structure

and function. These methods have been particularly useful in characterizing structural dynamics of different membrane proteins.


Related Publications

Molecular Dynamics Simulations of KcsA (ph gated K+ channel) (pdf)

Homology Modelling & Molecular Dynamics Simulations of Kir (ATP-gated K+ channel) (pdf)

Homology Modelling & Molecular Dynamis Simulations of GluR0 (Glutamate Receptor) (pdf)

Selectivity in K+ Channels (pdf)

Motions in K+ channels (pdf)

Molecular Modelling of KvAP (voltage gated K+ channel) (pdf)

Molecular Modelling of Shaker (voltage gated K+ channel) (pdf)

Coarse-grained simulations of K+ channels (pdf)

For movies on gating mechanism of KcsA (click here)



Current Research Projects

Glutamate Transporters

Accumulation of glutamate at extracellular (EC) space above physiological (micromolar) levels may cause neurotoxic effects. The

concentration of glutamate in the chemical synapse may increase by 103-104 fold succeeding its release triggered by an action potential,

and it is critical to have a mechanism in place to clear or shuttle the excess glutamate. This is where the glutamate transporters (GluT)

come into play. GluTs are trimeric membrane proteins located on neurons and glia (astrocytes). Their precise functioning (uptake

and re-uptake of glutamate) is essential for regulating the cycle of signaling, or terminating the synaptic transmission. They not only

maintain the glutamate concentration below excitotoxic levels but also regulate the activity of the glutamate receptors in the synapse:

they prevent sustained activation and thereby 'desensitization' of ionotropic receptors, and influence glutamatergic transmission by

metabotropic receptors. GluTs also assists in regulating the glutamate-glutamine cycle, i.e. conversion into glutamine in the glia,

succeeded by the transport to neurons to be converted back to glutamate.


Related Publications

Mechanism of extracellular gate opening in outward facing GltpH (pdf)

Mechanism of substrate release in inwards facing GltpH (pdf)

Large Scale motions regulate functional properties of GltpH (pdf)

For movies and more information on Glutamate Transporter simulations (click here)


Glutamate Receptors

Ionotropic glutamate receptors harbor two domains in their extracellular region, the membrane-proximal ligand-binding domain (LBD)

and the distal N-terminal domain (NTD). These are involved in signal sensing: the LBD binds L-glutamate, which activates the receptor

channel. Ligand-binding to the NTD modulates channel function in NMDA receptors, which has not been observed for AMPA receptor (AMPAR) NTDs.

Structural data suggest that AMPAR NTDs are packed into tight dimers and have lost their signaling potential. We have characterized NTD dynamics

from both AMPAR- and NMDAR- subfamilies using a variety of computational tools leading to a description of the motions which underly NMDAR NTD allosteric

signaling. Unexpectedly, AMPAR NTDs are capable of undergoing a similar dynamic spectrum; although dimerization imposes restrictions the two subfamilies

sample similar,interconvertible conformational subspaces. Finally, we solve the crystal structure of the AMPAR GluA4 NTD, combined with all-atom molecular

dynamics simulations we characterize regions pivotal for an as yet unexplored dynamic spectrum of AMPAR NTDs.


Related Publications

Dynamics and allostery in N-terminal domain of iGLuR2 (pdf)

Comparison of dynamics of NMDA- and AMPA N-terminal domains (pdf)

MD movie of NTD-interface destabilization


HIV-glycoprotein gp120

Infection of HIV-1 begins with a series of dynamic binding events between the trimeric glycoprotein envelope spike and the host cell

CD4 and chemokine receptors. The envelope trimer (gp160) is composed of three gp120 glycoproteins and the three transmembrane gp41 proteins.

The first dynamic event occurs via binding of gp120 to the host T-cell CD4 receptor, followed by extensive restructuring of gp120.

This conformational change results in the exposure of the chemokine binding site on gp120, thus permitting binding to either of the chemokine

receptors. Upon chemokine receptor binding a second conformational change occurs in the gp41 to form the fusion peptide that inserts in the host

cell membrane, leading to viral entry. The CD4 induced gp120 conformational change has been characterized thermodynamically, showing a highly

favorable binding enthalpy balanced with a highly unfavorable molecular ordering. This thermodynamic signature resembles protein folding, rather

than binding, and reflects the large molecular ordering of gp120 upon CD4 binding. A similar thermodynamic signature is exhibited by soluble

CD4 (sCD4) binding to both full-length gp120 (gp120full) and the truncated core gp120 (gp120core).


Related Publications

Communication propensities in gp120 (pdf)

Enhanced dynamics upon ligand binding in gp120 (pdf)

Spontaneous rearrangement of Beta strands 20/21 in unliganded gp120 (pdf)

MD movie of gp120 Spontaneous Rearrangement



Pseudomonas aeruginosa (PA) is an opportunistic pathogen that contributes to the mortality of patients with cystic fibrosis (CF), burn victims

and immune-compromised individuals. The virulence of PA is mediated by the secretion of virulence factors such as repeat-of-toxin(RTX)

proteins. Alkaline protease (AprA), one such RTX-protein secreted by PA is implicated in multiple modes of bacterial infection. The protease

is secreted in an unfolded form into the extracellular space, where it undergoes transition to a functional, folded form. The disorder-to-order

transition of the RTX-domain has been shown to be mediated by Ca2+ binding and the folded RTX-domain itself is proposed to facilitate proteolytic

domain (PD) folding, via the PD/RTX-domain interface. The underlying detailed mechanism of the toxic activities of RTX-toxins remains to be

elucidated. What is known however is that the intrinsic disorder of the RTX-domain in the bacterial cytosol facilitates secretion and that the

unfolded form of the RTX-toxin limits the enzymatic activity. The identity of structural elements of the protease which are required for regulation

of protein folding is a topic of investigation. These studies are aimed at identification of specific structural elements that stabilizing the protein

fold. This information can be used to design exepriments to characterize structure and function of misfolded proteins.


HSV-1 glycoprotein gH1

Over the last decade and a half, herpes simplex virus type 1 (HSV-1) has been developed into a leading gene therapy vector system.

Its large double-stranded DNA genome can simultaneously accommodate multiple therapeutic genes and the virus has broad cell and tissue

tropism, enabling its application to many different diseases. Recent advances have produced HSV-1 vectors that are limited in their infectivity

to one or a few cell types, advancing the prospect that invasive vector injection at the disease site can be replaced with systemic application.

Our goal is to produce viruses that are optimally infectious for their specific target cells or organs to increase their therapeutic efficacy

on systemic delivery. To this end we have isolated gain-of-function mutations in several of the HSV-1 glycoproteins that act in concert to

mediate virus entry into cells, gD, gB, gH and gL. We seek to understand the structural basis of the entry-enhancing effects

of mutations in gH using the published structures of gH from related herpesviruses, particularly HSV-2, for guidance. An understanding of the

structural consequences of these mutations will facilitate the rational engineering of gH and its direct partners in entry, gD and gB, to

accomplish further gains in entry efficiency. These studies promise to provide novel information about the key interactions between

the HSV-1 entry glycoproteins, which may lead to the design of new and highly specific anti-viral drugs.