Structure and Dynamics of Transport L8-10 (Chris Dempsey) Flashcards
Does the cytoplasm have a high sodium or potassium conc?
High intracellular K+ conc
Low intracellular Na+ conc
Name three types of energising transporters?
energetically unfavourable
Coupled transporter
ATP-driven pump
Redox-driven pump
What is coupled transport known as?
Secondary transport
Primary transport uses ATP
What gradient do Euks and Proks mainly use?
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)
What is the equation for energy available from the PMF?
ΔG(H+in-out) = F.ΔΨ - 2.303RT.ΔpH
Units J.mol-1
What type of transporter is lac permease (LacY)?
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
MFS members are responsible for what?
Nutrient uptake
Signal Transduction
Drug and noxious compound extrusion
In lactose permease how many amino acids are irreplaceable for active transport?
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
Structure of lactose permease
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
H+ transport in lactose permease
Outward-H+ protonates E269
Inward-H+ protonates E325/H322. E269 bonded to R144
OUTWARD
R144 binds E126
Feature of GLUT1 (mammalian transporter)
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.
In GLUT1 where are the regions where disease related mutations can occur?
1) substrate binding site
2) TM domain ICH interface
3) residues lining transport path
Sodium potassium pump
3Na+ out
2K+ in
ATP linked pump
How many members of solute sodium symporters indentified so far? Structure?
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
Example of SSS? solute sodium symporter?
Glalactose transporter
Example of antiporter?
Na+/Ca2+ antiporter
Uses sodium gradient into cells to pump Ca2+ out of cells
3Na+ in/ 1Ca2+ out
10TM helix - 2x5 TM inverted repeats
Three types of ATP-driven primary transporters
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
ABC transporters
48 in humans
80 in E.coli
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
NBDs
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
Mitochondrial transporters
each transporter recognises 2 molecules (often similar structure)
Can be uni/sym/antiporters
All have conserved protein fold
SIX TM HELICES
Mitochondrial ADP/ATP carrier
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
Mitochondrial phosphate carrier
symporter driven by pH gradient
No net charge H+ and H2PO4- are transported together
Electroneutral
ATP/ADP carrier and Pi carrier
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
Multidrug resistance
What is the most prevalent drug transporter?
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
Structure of P-glycoprotein
ABC transporter
12 TM helices make up TMD
single polypeptide chain
Resistance, Nodulation, Division (RND) family
Tripartite multidrug efflux pumps
Structure
Exporter found in bacteria
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
SMR (small multidrug resistance) transporters
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
