Notes from our 10.22.01 meeting with Irmi
FtsY is composed of the A domain (~200 residues) and the NG domain (~300 residues). The A domain is never ordered and a structure including this domain has not been obtained. Apo forms have been tried to be crystallized without success. Deleting 49 residues or 100 residues of the A domain leads to 2.8 Angstrom crystal structures in which the A domain is disordered. There is a conserved salt link and turn between the A and N domains. The A domain should be a-helical but alone is not stable. They may try to just look at this domain itself or with a small protein construct by NMR CD.
With Irmi's A. ambivalens, high salt must be used to keep the protein in solution and because of the salt, the protein will not bind the GTP nucleotide. The pI of FtsY is 4.1 and it is always a monomer when crystallized. There is a chain of Glu residues at the N-domain. Irmi's group is now working on solving the apo Ffh structure from Sulfolobus sp.
Different species of SRP and SRP receptor equals different inactivation.
Arg residues are catalytically important for both SRP and SR GTP hydrolysis. Irmi is mutating Arg to Ala as in Ras (may not see any effect mutating to Lys). The Arg191 proposed flip in the Freymann paper for the apo versus GMPPNP structure could be catalytically important as an arginine finger.
The Gln61 mutation in Ffh renders the enzyme inactive.
FtsY (SR) and Ffh form a complex when both are bound to GTP. When the SR/SRP/ribosome/nascent protein complex reaches the cell membrane (Prok.), the hydrolysis of FtsY is stimulated. This membrane association induces a high turnover rate of the enzyme. FtsY then acts as a GAP (guanine nucleotide activating protein) for Ffh, which hydrolyzes its GTP.
The charge distribution differs for the SRP and its receptor. The SRP is always bound with its RNA.
E. coli bacterial SRP and receptor do not work with those from the mammalian cell: no complex forms between FtsY (bacterial receptor) and SRP54 (mammalian SRP).
FtsY and Ffh should form a complex in which the GTP binding domains come together when both proteins are bound to GTP. The looks like a C2V relationship where the GTP binding domains pair but the M domain in Ffh and the A domain in FtsY are opposite each other.
The presence of a signal sequence on a nascent chain increases the binding of SRP to the ribosome. In the presence of GTP, the SRP receptor binds to the SRP and causes the SRP to dissociate from the signal sequence and the ribosome. As Ffh and FtsY form a complex the GTPases are activated or they both bind GTP and them form a complex? Form complex, induces conformational change, then bind GTP
Can compare the ATP binding proteins or MutS or the 1NRC (Irmi used) to the GTP binding proteins such as the SRP.
Liz Sauer-Eriksson (now at Umea U in Sweden) is working on the Mycoplasma FtsY and has a structure both with and without SO4. Without the salt, there is a change in the local conformation of the loops. The SO4 binds in the active site. Any structure with this in the active site (as Walter's is) should not be considered a true apo- form. In Liz's SO4 structure, the P loops close.
Peter Walter claims (but Irmi disagrees) that there is a Arg191 (motif III)-Asp135 (motif II) salt bridge in the T. Aquaticus Ffh that is present in the apo form but disrupted in the Mg2+GDP bound form.
Stephen Cusack collaborates with Irmi and is working on the human SRP.
Eitan Bibi (Weizmann, Israel) (PNAS. (2000) 97:4621-4626.) has confirmed that membrane targeting of E. coli ribosomes requires the prokaryotic SRP receptor FtsY in vivo and that depletion of Ffh has no significant effect on the binding of ribosomes to the membrane although depletion of Ffh is detrimental to growth. The SRP does not play the critical role of targeting ribosomes to the membrane. Their hypothesis is that FtsY may mediate ribosome targeting in the absence of the SRP.
Joen Luirink has reconstituted liposomes to follow GTP hydrolyzed by FtsY. In the intact cell the hydrolysis differs from the liposome which raises the question of the involvement of another protein.
Kevin Weeks (UNC Chapel Hill) is collaborating with Irmi and footprinting the NG domain of the SRP with the RNA.