structural biology of membrane proteins problems of structure determination & membrane-specific...

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.