structural biology of membrane proteins problems of structure determination & membrane-specific...
TRANSCRIPT
Structural Biology of Membrane Proteins
• Problems of structure determination &
• Membrane-specific solutions• KcsA structure• Mechanistic insights• KvAP and voltage sensing
• Shaker (KV1.2)
Key Difficulties in MP Structure
• Available amounts….expression systems?
• Solubility and solubilization; detergents
• Microheterogeneity; PTMs?• Mutational alteration to
facilitate crystallization; affinity tags
• Homologs; genomics
Channel Structures Provide..
• Details of structure• Familial relationships• Structural basis of ion selectivity• Mechanism of ion dehydration by
channel• Linkage between pore and
voltage sensor (for VS channels); gating mechanisms???
Genomics and Channel Structures
VSKC homolog (kcsA) discovered in S. lividans as ORF
Monomer MW ~18kD
Hydropathy analysis predicts 2 TM domains
32% identity to shaker in “signature” sequence
KvAP
What Defines a K+ Channel?
• Founder of VS cation channel family • Widely distributed in nature; multiple
isoforms in most organisms• Monomer MW ~70kD (eukaryotes)• Activated by membrane depolarization• Most isoforms have 6 predicted tm
domains/monomer; functional tetramers
• Mutagenic analysis of pore blockers: pore between S5 and S6
Schrempf, et al (1995) “A prokaryotic potassium ion channel with two predicted transmembrane segments from S. lividans,” EMBO J 14, 5170.
Cortes & Perozo (1997) “Structural dynamics of the S. lividans K channel: oligomeric stoichiometry and stability,” Biochemistry 36, 10343.
Doyle et al (1998) “The structure of the potassium channel: molecular basis of K-conduction and selectivity,” Science 280, 69.
KcsA: The 1st Structure
VSKC Architecture?
Sequences of K-channel Pore Regions
Genomic Identification of KcsA
Signature region
Is KcsA a Channel?
Single channel analysis
Current-voltage relationship
KcsA Oligomerizes in Detergent
stain
western
1. Cell extract2. Cell super3. Membranes4. DDM extract5. NTA-purified DDM extract
KcsA Tetramers Maintained in DDM
Structural Organization of KcsA
KcsA Tetramer Defines Central Pore
K+
Polarity of KcsA Pore Region
Blue, cationic; red, anionic; yellow, hydrophobic
Aromatic Clustering at KcsA Membrane Interface
Proposed Mechanism of Ion Translocation
Lake Kalemia: note charge stabilization by helix dipoles
Ionic Stabilization by Helix Dipole in ClC Channels
Comparison of KcsA and ClC Pores
ClC
KcsA
How Does Voltage-sensing Work?
SCAM analyses consistent with transverse S4 movement
KvAP: Structure and a Proposed Mechanism for Voltage Sensing.
Jiang, MacKinnon et al (2003) “X-ray structure of a voltage-dependent K-channel,” Nature 423, 33-41; “The principle of gating charge movement in a voltage-dependent K channel,” Nature 423, 42-48.
The KvAP Bombshell
Sequence Homology in Voltage-dependent K-
channels
KvAP: a Voltage-sensitive Channel
Backbone Structure of the KvAP Tetramer
KvAP: The Side View
Putative voltage sensor paddle
Architecture of KvAP Monomer
Proposed Paddle Movement during Gating
Questions about the KvAP Structure
• Validity of KvAP as paradigm?• Solubilization?• Modification?• Crystallization aids?• Impact of any/all on overall
structure?
Science, 309: 897-908
Shaker and KvAP: Not quite the Same Beast
Accessory Subunits in K-channels
K-channel Pores Are Conserved
Structure of the shaker Tetramer
Backbone Comparison: KvAP vs shaker
Pore Communication with Voltage Sensor in Kv
Channel
The Kv1.2 Tetramer: note that the nearest neighbor of S4 in the red subunit is S5 from the blue subunit
Closeup view showing the weak interaction between S4 and the pore region
Revised View of Voltage-sensing
Note S6 changes between open and closed states.