El lunes y martes 20 y 21 de noviembre coordinamos un simposio internacional en la Fundación Ramón Areces, sobre los defectos del transporte de aminoácidos.
Engler and Prantl system of classification in plant taxonomy
Edmund Kunjii Medical Research Council. Mitochondrial Biology Unit. Cambridge.
1. Mitochondrial carriers – introduction and structure
mitochondrial aspartate/glutamate carrier
Fiona Fitzpatrick Antoniya Aleksandrova (NIH)
Daniel Jones
Martin King
Vasso Mavridou
Jonathan Ruprecht
Tom Schirris
Sotiria Tavoulari
Chancievan Thangaratnarajah
Shane Palmer
2. The mitochondrial carrier family
Palmieri, F. (2013) The mitochondrial transporter family SLC25: identification, properties and
physiopathology. Mol Aspects Med 34, 465-484
4. Topology model of the ADP/ATP carrier
PX[DE]XX[KR]
Saraste and Walker, 1982Klingenberg et al., 1982
5. The 5-Å projection structure of the yeast ADP/ATP carrier Aac3p
Kunji ERS, Harding M (2003) J. Biol. Chem. 278:36985-36988.
Structure is three-fold pseudo-symmetrical with six transmembrane α-helices
Translocation pathway for the substrate is through the centre of the molecule
Transporter is monomeric, questioning dimeric models for the mechanism
6. Electron crystallography.
Kunji ERS, Harding M (2003) J. Biol. Chem. 278:36985-36988.
X-ray crystallography.
Ruprecht JJ, Hellawell AM, Harding M, Crichton PG, Mccoy AJ, and Kunji ERS. (2014) Proc. Natl. Acad. Sci. U.S.A
111, E426-E434.
Size exclusion chromatography in the alkyl-maltoside and cymal detergent series.
Bamber L, Harding M, Butler PJ, Kunji ERS (2006) Proc. Natl. Acad. Sci. U.S.A. 103:16224-16229.
Kunji ERS, Harding M, Butler PJ, Akamine P (2008). Methods 46:62-72
Sedimentation equilibrium analytical ultracentrifugation.
Bamber L, Harding M, Butler PJ, Kunji ERS (2006) Proc. Natl. Acad. Sci. U.S.A. 103:16224-16229.
Differential tagging and affinity chromatography.
Bamber L, Slotboom DJ, Kunji ERS (2007) J. Mol. Biol. 371:388-395.
Blue native gel electrophoresis
Crichton, PG, Harding, M, Ruprecht, JJ, Lee, Y, Kunji, ERS (2013), J. Biol. Chem. 288:22163-22173
Co-expression of wild-type and cysteine-less ADP/ATP carriers.
Purification and reconstitution of wild-type and cysteine-less ADP/ATP carriers.
Bamber L, Harding M, Monné M, Slotboom DJ, Kunji ERS (2007) Proc. Natl. Acad. Sci. U.S.A. 104:10830-10834.
Two networks explain strict exchange without the need for invoking a dimer.
Mechanism is three-fold symmetric.
A conserved and asymmetric interface is not present
Robinson AJ, Overy C, Kunji ERS (2008) Proc. Natl. Acad. Sci. U.S.A. 105:17766-17771.
Mitochondrial carriers are monomeric in structure and function
7. The structure of the bovine ADP/ATP carrier (1OKC)
inhibited by carboxy-atractyloside
H1
H2
H3
H4
H6
H5
H1
H2
H3
H4
H6
H5
h12
h34h56
h12
h34h56
mitochondrialinner
membrane
matrix
Pebay-Peyroula et al., 2003
8. Architecture of the yeast mitochondrial ADP/ATP carriers
Jonathan Ruprecht
mitochondrialinner
membrane
Mitochondrial
matrix
intermembrane
space
lateral view cytoplasmic view
S
P
P
SPP
11. The mitochondrial aspartate/glutamate carrier is a chimera
Palmieri L, Pardo B, Lasorsa FM, del Arco A, Kobayashi K, Iijima M, Runswick MJ, Walker JE,
Saheki T, Satrústegui J, Palmieri F, (2001) EMBO J. 20, 5060-5069.
12. Oligomeric state of the human aspartate-glutamate carrier
Chancievan Thangaratnarajah
twice 74 kDa > it is a dimer!
14. Architecture of the regulatory domain 1
Chancievan Thangaratnarajah Jonathan Ruprecht
EF1
EF2
EF3
EF4 EF5
EF6
EF7
EF8
EF2
C-terminal
helix
Lateral view
15. Architecture of the regulatory domain 2
Chancievan Thangaratnarajah Jonathan Ruprecht
EF1
EF2
EF3
EF4 – EF8
C-terminal helix
Cytoplasmic view
16. Binding pocket of the C-terminal helix is conserved
Chancievan Thangaratnarajah and Jonathan Ruprecht
17. Conformational changes of the regulatory domain of the
human mitochondrial aspartate/glutamate carrier
Chancievan Thangaratnarajah Jonathan Ruprecht
18. Missense mutations in the regulatory domain leading to citrin deficiency
Chancievan Thangaratnarajah
19. calcium-free
state
calcium binds to
EF-hand 2 in the mobile unit
mobile unit
opens the vestibule
C-terminal domain
binds to the hydrophobic groove
carrier domain
access to the carrier domain
substrate can be translocated
calcium
dissociates
C-terminal domain dissociates
mobile unit closes the vestibule
calcium free state
access closed by the vestibule
matrix
regulatory domain
carrier domain
intermembrane space
inner membrane
Proposed mechanism for calcium-regulation
Chancievan Thangaratnarajah and Jonathan Ruprecht
34. Interaction energies of substrate binding and salt bridge
formation have to be equal Roger Springett and Edmund Kunji
“Consequently, the model predicts that there will be other interactions in addition to those of
the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier“
36. Atomic structures of yeast ADP/ATP carriers Aac2p and Aac3p
Jonathan Ruprecht
Aac2p Aac3p
37. Structural evidence for a domain-based transport mechanism
Jonathan Ruprecht
domain 1 domain 2 domain 3
38. The inter-domain interfaces are dynamic
Jonathan Ruprecht and Edmund Kunji
odd-numbered helices even-numbered helices
GxxxG
motif
39. Domain motions for formation of the cytoplasmic network
cytoplasmic state matrix state
40. Mitochondrial carriers provide the transport steps of amino acids, keto acids, fatty acids, nucleotides,
co-factors and inorganic ions across the inner membrane of mitochondria.
The structures are three-fold pseudo-symmetric and form a functional monomer.
A single site for binding of substrates and protons is present in the central cavity, corresponding
approximately to the middle of the membrane.
Ion pairs on the odd-numbered α-helices form the matrix network and ion pairs on the even numbered
α-helices form the cytoplasmic network.
Opening and closing of the carrier induced by substrate binding is coupled to alternating disruption and
formation of the networks via a 3-fold rotary twist of the domains.
The interaction energies of the networks explain the transport mode, i.e. strict equimolar exchange
versus net import.
The mitochondrial aspartate/glutamate carrier is dimeric via dimerisation of the regulatory domain.
Regulation by calcium of AGC might occur by opening and closing access to the carrier domain
The proposed mechanism explains mutations observed in diseases caused by dysfunctional carriers
Conclusions