Joint UCT/UWC Masters Programme in Structural Biology
Module: Structural basis of ion transport through membranes
David B McIntosh, Clinical Laboratory Sciences
Assignments:
1. Detail the nature of the forces that a K+ ion is subjected to as it
traverses a K+ channel. (to be handed in Thu 25 Sept)
2. Describe the path in atomic detail that a H+ follows during passage
through bacteriorhodopsin. Provide reasons at each critical point why it
goes in an extracellular direction rather than intracellular. (to be handed
in Fri 3)
Mon 15 Sept: 9.00 am. Introductory lecture – The problem of getting ions
across a hydrophobic barrier and nature’s solutions.
Tue 16 Sept: Facilitated diffusion through biomembranes – prepare paper by
Doyle, AD, Cabral, JM., Pfuetzner, RA., Kuo, A., Gulbis, JM., Cohen, SL.,
Chait, BT., and MacKinnon R. “The structure of the potassium channel:
Molecular basis of K+ conduction and selectivity”, Science 280, 69-77 (1998).
PDB accession code: 1BL8
Lab work:
a. Follow the structure residue by residue from the N-terminal end,
making notes on the how the secondary structure is held in its tertiary
and quaternary structures. What are the weak points, what are the
strong? What is stabilising the structure as it is? Could the selectivity
filter collapse if K+ ions were absent? Where are possible places that
the channel could be blocked? Are there agents bind at these points?
b. Note the location of the tryptophan residues pointing into the lipid
bilayer. What is perculiar? Can you locate the surfaces of the lipid
bilayer?
Wed 17 Sept: Details of the selectivity filter – prepare papers by
Morais-Cabral et al “Energetic optimisation of ion conduction rate by the K +
selectivity filter”, Nature 414, 37-42 (2001).
PDB accession codes: 1JVM, 1K4C, and 1K4D
Zhou et al “Chemistry of ion coordination and hydration revealed by a K +
channel-Fab complex at 2.0 Å resolution” Nature 414, 43-48 (2001).
Roux, B., and MacKinnon, R. “The cavity and pore helices in the KcsA K +
channel: Electrostatic stabilization of monovalent cations” Science 285, 100102
Labwork:
a. View structures and examine the position and coordination of ions
b. Locate Asp-80 and Glu-71
c. See if you can see the pinching in of the selectivity filter at Gly-77 in
1K4D
Thu 18 Sept: Structural basis of gating – prepare paper by
Kuo, A., Gulbis, JM., Antcliff, JF., Rahman, T., Lowe, ED., Zimmer, J.,
Cuthbertson, J., Ashcroft, FM., Ezaki, T., Doyle, DA. “Crystal structure of the
potassium channel KirBac1.1 in the closed state” Science 300, 1922-1926
(2003).
PDB accession code: 1P7B
Lab work:
a. Examine the N-terminal linker and Cterminal domains. Locate the circles
of negatively and positively charged
residues in the C-terminal domains.
What are their roles? Check out the
Phe-146. Verify that the inner helix
is bent at Gly-134. How are the
selectivity filter and pore helices
changed from the KscA structure?
Significance?
b. Write notes on the gating hypothesis.
Fri 19 Sept: Introductory lecture: Bacteriorhodopsin - a light powered H+
pump
Thu 25 Sept: The ground state structure – prepare paper by
Luecke, H., Schobert, B., Richter, H. T., Cartailler, J-P., Lanyi, JK. “Structure
of bacteriorhodopsin at 1.55 Å resolution” J. Mol. Biol. 291, 899-911 (1999)
PDB accession code: 1C3W
Lab work:
a. Get familiar with the layout
of the 7 helices. See
where the phospholipids
are located and the
position of aromatic
residues relative to them.
b. Trace the conductance
pathway and locate the
key residues. Examine in
detail the organisation of
water molecules under the
Schiff base and towards
the extracellular surface.
Go through the text of the
paper with the structure in
front of you.
Fri 26 Sept: Evolution of activated states – prepare papers by
Luecke, H., Schobert, B., Richter, H-T., Cartailler, J-P., and Lanyi, JK.
“Structural changes in bacteriorhodopsin during ion transport at 2 Angstrom
resolution” Science, 286, 255-260 (1999)
PDB accession code: 1CBR and
1C8S (BR and M states of mutant
D96N)
Lab work:
a. In the ground state
structure verify that the
mutation does not change
the organisation of
residues and water
molecules below the Schiff
base. Check the
downward orientation of
the Schiff base.
b. In the M intermediate
check the upward
orientation of the Schiff
base, the single water
molecule around Asp-85 and 212. See the downward orientation of
Arg-82 and its relationship to Glu-194 and 204. Understand how this
allows for the ionisation of the latter.
c. Examine the channel above the Schiff base, note the lack of water
molecules (just two some distance away). Why is the channel so
dehydrated?
d. How have the helices changed from the BR state? Concentrate on
helices F and G.
Lanyi, JK., and Schobert, B. “Mechanism of proton transport in
bacteriorhodopsin from crystallographic structures of the K, L, M1, M2, and M2’
intermediates of the photocycle” J. Mol. Biol. 328, 439-450 (2003)
PDB accession code: 1O0A (L intermediate)
Lab work:
a. Colour the BR and L proteins and water molecules differently. Check
that the 13 trans to cis isomerisation has taken place, the location of
the protonated Schiff base, the water molecules, and the fact that Arg82 has not moved from the BR state.
b. Note how helix C has bent inwards.
Schobert, B., Brown, LS., and Lanyi, JK. “Crystallographic structures of the M
and N intermediates of bacteriorhodopsin: Assembly of a hydrogen-bonded
chain of water molecules between Asp-96 and the retinal Schiff base” J. Mol.
Biol. 330, 553-570 (2003)
PDB accession codes:1P8U
(N’ intermediate)
Lab work:
b Again colour the BR
and N’ proteins and
water molecules
differently. Check the
hydration of the region
above the Schiff base.
Mon 29 Sept: Overview of conformational changes and mechanism of pKa
changes - prepare paper by
Sampogna and Honig “Environmental effects on the protonation states of
active site residues in bacteriorhodopsin” Biophys J. 66, 1341-1352 (1994)
Tue 30 Sept: Introductory
lecture: Ca2+-ATPase – An
ATP driven Ca2+ pump
Wed 1 Sept: The E1. Ca2
structure – prepare paper by
Toyoshima, C., Nakasako,
M., Nomura, H., and Ogawa,
H. “Crystal structure of the
calcium pump of
sarcoplasmic reticulum at
2.6 Å resolution” Nature 405,
647-655 (2000)
PDB accession code: 1EUL
Lab Work:
a Become familiar with
each domain
structure. Locate
Asp-351 the residue
phosphorylated
during the catalytic
cycle and note the
winding of the highly
conserved motif
351DKTGTLT around and behind it. Also find highly conserved motifs
625TGD, 701TGDGVND, and 684K. Speculate on their possible roles.
b Find the two Ca2+ and establish how they are ligated. Notice how one
ion is coordinated through unravelling of part of a transmembrane helix.
The binding and release of Ca2+ from the top (cytoplasm) is ordered.
Can you see why?
c Locate Phe-487 and Leu-562 in the nucleotide binding domain. The
adenine of ATP probably fits between these two residues. Check out
Lys-515 which is specifically labelled with FITC. Look for Lys-492
which is specifically photolabelled by TNP-8N3-ATP. Find Arg-678 in
the P-domain. Lys-492 and Arg-678 are cross-linked by
glutaraldehyde. It is thought to be a zero distance cross-linkage and
provides a clue how the N-domain must fit with the P-domain. How
close do you reckon the N-domain must approach Asp-351 for
phosphorylation to occur.
d Locate conserved motif 179TGES in the actuator or A domain.
e Try and imagine how phosphorylation of Asp-351 could drive changes
at the transport sites.
Thu 2 Oct: Structural changes induced by Ca2+ binding – prepare paper by
Toyoshima, C., and Nomura, H. “Structural changes in the calcium pump
accompanying the dissociation of calcium”. Nature 418, 605-611 (2001)
PDB accession code: 1IWO
Lab work:
a Note the huge change in
domain organisation. In
particular see where conserved
motif 179TGES of the A domain
is. Note how much closer the
nucleotide site is to Asp-351.
b Look what has happened to the
membrane helices and Ca2+
sites.
Fri 3 Oct: Electrostatic fireworks in the engine room – prepare papers by
McIntosh, DB., Woolley, DG., Vilsen, B., and Andersen, JP. “Mutagenesis of
segment 487Phe-Ser-Arg-Asp-Arg-Lys492 of sarcoplasmic reticulum Ca2+ATPase produces pumps defective in ATP binding” J. Biol. Chem. 271,
25778-25789 (1996)
McIntosh, DB., Woolley, DG., MacLennan, DH., Vilsen, B., and Andersen, JP.
“Interaction of nucleotides with Asp351 and the conserved phosphorylation loop
of sarcoplasmic reticulum Ca2+-ATPase” J. Biol. Chem. 274, 25227-25236
(1999)