Structural Biology of Membrane Proteins

1
Structural Biology of Membrane Proteins

• Problems of structure determination
• Membrane-specific solutions
• KcsA structure
• Mechanistic insights
• KvAP and voltage sensing
• Shaker (KV1.2)

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

3
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???

4
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
5
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

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KcsA The 1st Structure
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.
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VSKC Architecture?
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Sequences of K-channel Pore Regions
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Genomic Identification of KcsA
Signature region
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Is KcsA a Channel?
Single channel analysis
Current-voltage relationship
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KcsA Oligomerizes in Detergent
1. Cell extract 2. Cell super 3. Membranes 4. DDM
extract 5. NTA-purified DDM extract
stain
western
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KcsA Tetramers Maintained in DDM
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Structural Organization of KcsA
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KcsA Tetramer Defines Central Pore
K
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Polarity of KcsA Pore Region
Blue, cationic red, anionic yellow, hydrophobic
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Aromatic Clustering at KcsA Membrane Interface
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Proposed Mechanism of Ion Translocation
Lake Kalemia note charge stabilization by helix
dipoles
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Ionic Stabilization by Helix Dipole in ClC
Channels
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Comparison of KcsA and ClC Pores
ClC KcsA
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How Does Voltage-sensing Work?
SCAM analyses consistent with transverse S4
movement
21
The KvAP Bombshell
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.
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Sequence Homology in Voltage-dependent K-channels
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KvAP a Voltage-sensitive Channel
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Backbone Structure of the KvAP Tetramer
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KvAP The Side View
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Architecture of KvAP Monomer
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Proposed Paddle Movement during Gating
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Questions about the KvAP Structure

• Validity of KvAP as paradigm?
• Solubilization?
• Modification?
• Crystallization aids?
• Impact of any/all on overall structure?

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Science, 309 897-908
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Shaker and KvAP Not quite the Same Beast
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Accessory Subunits in K-channels
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K-channel Pores Are Conserved
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Structure of the shaker Tetramer
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Backbone Comparison KvAP vs shaker
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Pore Communication with Voltage Sensor in Kv
Channel
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Revised View of Voltage-sensing