Structure and Dynamics of Transport L8-10 (Chris Dempsey)

Question 1

Does the cytoplasm have a high sodium or potassium conc?

Answer 1

High intracellular K+ conc

Low intracellular Na+ conc

Question 2

Name three types of energising transporters?

energetically unfavourable

Answer 2

Coupled transporter
ATP-driven pump
Redox-driven pump

Question 3

What is coupled transport known as?

Answer 3

Secondary transport

Primary transport uses ATP

Question 4

What gradient do Euks and Proks mainly use?

Answer 4

Euk p.memb-mainly uses Na+ gradient since low intracellular Na+ (source of free energy coupled to transport process)
Prok p.memb-mostly use H+ gradient (PMF)

Question 5

What is the equation for energy available from the PMF?

Answer 5

ΔG(H+in-out) = F.ΔΨ - 2.303RT.ΔpH

Units J.mol-1

Question 6

What type of transporter is lac permease (LacY)?

Answer 6

Proton linked secondary symporter
Transport is electrogenic
It is part of the Major facilitator super-family (MFS)
10,000 members, transport solutes, passive or secondary active transport

Question 7

MFS members are responsible for what?

Answer 7

Nutrient uptake
Signal Transduction
Drug and noxious compound extrusion

Question 8

In lactose permease how many amino acids are irreplaceable for active transport?

Answer 8

6
ALL charged 
E126
R144
E269
R302
H322
E325 (can bind the proton)
4 of these lie b/w the substrate binding pocket
W151 forms a non polar interaction with pyranose ring of galactose

Question 9

Structure of lactose permease

Answer 9

Two 6TM repeats (composed of two 3 TM repeats)

P and G residues (proline and glycine) destabilise helices/induce helix kinks-relation to conformational felxibility

Question 10

H+ transport in lactose permease

Answer 10

Outward-H+ protonates E269
Inward-H+ protonates E325/H322. E269 bonded to R144

OUTWARD
R144 binds E126

Question 11

Feature of GLUT1 (mammalian transporter)

Answer 11

ICH-intracellular helix bundle
XylE and GlcP also has ICH
ICH is likely to be feature of sugar transporters
May function as a latch to secure closure of intracellular gate in outward facing conf.

Asn residue in GLUT1 uniporter is equiv to Asp residue in other sugar transporters which play a critical role in proton coupling to H+ linked transport

GLUT1 uses glucose conc gradient for transport.

Question 12

In GLUT1 where are the regions where disease related mutations can occur?

Answer 12

1) substrate binding site
2) TM domain ICH interface
3) residues lining transport path

Question 13

Sodium potassium pump

Answer 13

3Na+ out
2K+ in
ATP linked pump

Question 14

How many members of solute sodium symporters indentified so far? Structure?

Answer 14

250
14TM helices with an “INVERTED REPEAT TOPOLOGY”
TM2-TM6 and TM7-TM11 are “upside down” in membrane with respect to each other
Emply “alternate access” model

Question 15

Example of SSS? solute sodium symporter?

Answer 15

Glalactose transporter

Question 16

Example of antiporter?

Answer 16

Na+/Ca2+ antiporter
Uses sodium gradient into cells to pump Ca2+ out of cells
3Na+ in/ 1Ca2+ out
10TM helix - 2x5 TM inverted repeats

Question 17

Three types of ATP-driven primary transporters

Answer 17

P-type pump (reversibly phosphorylated-changes direction of transport) transports ions ATP HYDROLYSIS
F-type (and V-type) proton pump ATP SYNTHESIS/HYDROLYSIS
ABC (ATP binding cassette) transporter-Pseudodimers ATP HYDROLYSIS

Question 18

ABC transporters

48 in humans
80 in E.coli

Answer 18

IMPORTERS 8-20TM helices
4 independent subunits
only in Proks
require a periplasmic binding protein to deliver the substrate to the transporter
Substrates for importers=nutrients
EXPORTERS 12TM helices
continuous polypeptide chain with NBDs (dimers)
Euks single polypeptide 4domains
Prok 2 diff subunits homodimer
found in both Proks and Euks
Multi drug resistance

Question 19

NBDs

Answer 19

share common fold and common seq motifs
WALKER A (p-loop)
WALKER B
2 ATP molecules bind at the INTERFACE of the 2NBDs and bring the 2 domains together (conf change)
Coupling helices move >10Å closer to ATP bound conf.
ATP binding is COOPERATIVE

C loop/ABC signature motif
LSGGW loves shagging got gonnorrhea whoops

Question 20

Mitochondrial transporters

Answer 20

each transporter recognises 2 molecules (often similar structure)
Can be uni/sym/antiporters
All have conserved protein fold
SIX TM HELICES

Question 21

Mitochondrial ADP/ATP carrier

Answer 21

Antiporter is driven by the membrane potential
electrogenic
ADP3-
ATP4-
takes one negative charge out of the mitochondrial matrix
energetically favourable
SIX TM HELICES (3x2helix repeats)
Pro/gly faciliate conf changes through flexing
helix kinks/felxibility from PRO/GLY
NB/ alpha helix doesn’t like having pralines and glycine

Question 22

Mitochondrial phosphate carrier

Answer 22

symporter driven by pH gradient
No net charge H+ and H2PO4- are transported together
Electroneutral

Question 23

ATP/ADP carrier and Pi carrier

Answer 23

ATP/ADP carrier - moves 1 charge but no protons
Pi carrier - mores 1H+ but no charge

ATP synthase uses 3-5H+ per ATP (depends on number of c subunits)
Overall transport of ADP/ATP/Pi uses up one H+ and one charge per ATP generated

Question 24

Multidrug resistance

What is the most prevalent drug transporter?

Answer 24

40% of all cancer cell lines develop resistance to cancer drugs - export of drugs by transporters
Most prevalent drug transporter= P-glycoprotein (P-gp) an ABC transporter (product of gene MDR-1)
Gene amplification of MDR-1 in many cancers causes overexertion of P-gp and resistance to a range of drugs
P-gp has a very broad specificity/polyspecificity - substrates range from 350-4000kDa (large and chemically diverse substrate pocket)

MDR proteins often encoded on plasmids-easily transferable b/w bacteria
SMR (small multidrug resistance) transporters
ABC multi drug resistance transporters
MFS multidrug resistance transporters

Question 25

Structure of P-glycoprotein

ABC transporter

Answer 25

12 TM helices make up TMD

single polypeptide chain

Question 26

Resistance, Nodulation, Division (RND) family
Tripartite multidrug efflux pumps
Structure
Exporter found in bacteria

Answer 26

Outer memb channel
Periplasmic adapter protein (links IM transporter to OM channel) anchored to IM by lipid moiety
Inner memb transporter (proton antiporter of the RND family)

broad substrate specificity

Question 27

SMR (small multidrug resistance) transporters

Answer 27

Proton drug antiprotons
Best characterised is EmrE
4TM helices INVERTED HOMODIMER in memb
Can be inserted in two different planes into the memb
broad substrate specificity