A pinboard by
Monika Kowalska

Postdoctoral student, University of Warsaw


My name is Monika Kowalska. I am a post-doctoral student at the Structural Biology Group, University of Warsaw, Poland, headed by Dr. Maria Górna. I am working on IFIT proteins (interferon induced proteins with tetratricopeptide repeats) which are effectors of the innate immune system. IFITs are expressed in infected cells and disrupt viral RNA by sequestration of transcripts, leading to inhibition of translation of viral proteins. The IFIT family is evolutionarily conserved in vertebrates and includes five human paralogues (IFIT1, 1B, 2, 3, 5). IFIT5 recognizes ssRNA with the triphosphate (PPP) group on the 5’end, whereas IFIT1 preferentially binds cap 0 groups on the 5’end of RNA. IFIT2 binds RNA regardless of the 5’ moiety. IFIT1 interacts with IFIT3, which is not known to bind any RNA. I am interested in the structure and function of IFIT1-IFIT3 complex. IFIT1 alone binds cap 0 RNA with nanomolar activity, but the complex IFIT1-IFIT3 binds RNA with much higher affinity (400-fold higher) than IFIT1 alone. I am trying to investigate how the two proteins interact with each other and what is the mechanism of enhanced RNA binding by the complex. To find it I am using several different methods, such as chemical crosslinking coupled with mass spectrometry, small angle x-ray scattering and microscale thermophoresis with wt and mutant IFIT1 and IFIT3. I also aim to test the activity of IFIT homologs from different organisms (fish, chicken, duck, rabbit) towards RNA binding (microscale thermophoresis, EMSA).


Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

Abstract: Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2′-deoxyuridine 5′-monophosphate (dUMP) and (3) 5-fluoro-dUMP (FdUMP) and N5,10-methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (“inactive” and “active,” respectively) in the loop 181–197, in each of the three mTS structures, the loop 175–191, homologous to hTS loop 181–197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein’s inability to adopt the inactive conformation. The binary structures of either dUMP- or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS–5-FdUMP–meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41–47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.

Pub.: 09 Sep '16, Pinned: 13 Jun '17