Page 70 - Annual report 2021-22

Annual Report 2021-22 |

Lipi Thukral

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Lipi Thukral works on computational structural biology to understand the interaction of proteins that
are associated with diseases. One of the focuses of her lab is to understand the interaction of proteins
with membranes. To this end, role of proteins that play essential role in autophagy, like LC3, was
studied.

Autophagy can be closely monitored through studying LC3 interaction on autophagosomal
membranes. However, the present understanding of LC3 structure and dynamics is based on cytosolic
(non-lipidated) protein. The dynamics of lipidated-Atg8 orthologs inserted in the membrane was
investigated. In silico phosphatidylethanolamine (PE) chain was parameterized in atomistic manner
and combined LC3-PE with heterogenous ER composition membranes. The rationale behind this
approach was to introduce multiple lipids that aid in autophagosome formation, in particular -
different charged interactions present in heterogenous lipid headgroups. Interestingly, LC3 molecules
form puncta that can be observed using fluorescence microscopy. The open question is how do LC3
proteins oligomerize. To address this, systems with multiple LC3s (>90) on a large ER membrane were
prepared. This essentially mimic the crowded environment of autophagosome wherein LC3
arrangement in oligomeric fashion on physiologically relevant membranes has been observed.
Spontaneous and stable formation of dimers was observed, and time-dependent investigation of
simulations showed that the protein has high tendency to oligomerize in higher order structures.
Analysis of interaction surface revealed novel interaction surface that may be critical for LC3. The same
was performed for all isoforms. At end, reliable structural models were built that mimic physiologically
relevant LC3 structures on simplistic membranes.

Another protein that plays an essential role in interaction with membranes and involved in cell-cell
communication is Notch. Notch is an evolutionarily conserved signaling pathway, which regulates cell
fate determination during embryogenesis and cellular homeostasis in adults via cell-to-cell
communication. This pathway derives its name from the receptor protein ‘Notch' located at the cell
membrane. Notch is a single pass transmembrane protein which interacts with its transmembrane
ligands, such as Delta-like protein (DLL) and Jagged (JAG) present on the neighbouring cells. Ligand
binding leads to proteolytic cleavages resulting in the release of the Notch intracellular domain (NICD)
into the nucleus, where it regulates the transcriptional complex to activate Notch target genes. Apart
from DLL and JAG, Notch has various binding partners that are known to work in a highly-context
dependent manner. Any impairment in Notch signaling pathway is known to cause various types of
diseases like cancer and rare genetic autosomal disorders like Alagille Syndrome, CADASIL (Cerebral
autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy), Adams-Oliver
Syndrome, etc.

An integrative assembly of Notch extra-cellular domain (NECD) was performed. The NECD is composed
of 1,752 amino acids and the structure for 10 out of 36 EGF-like repeats (EGF4-9 and EGF10-13) has
been resolved. In order to complete the N1 extracellular domain, a hybrid approach was followed that
included the modelling of EGF-like repeats based on the experimental input parameters and patching
them to the NRR with the TM. Since the interface between EGF9-10 has been suggested to be flexible,