X-Ray Crystallography and It

1
X-Ray CrystallographyandIts Applications

• By
• Bernard Fendler
• and
• Brad Groveman

2
Introduction

• Present basic concepts of protein structure
• Discuss why x-ray crystallography is used to
  determine protein structure
• Lead through x-ray diffraction experiments
• And present how to utilize experimental
  information to design structural models of
  proteins

3
Introduction to Protein Structure The
Crystallographers Problem

• What is the crystallographers problem?
  Structural Determination!
• Structure Function
• Amino acids are strung together on a carbon chain
  backbone.
• As a result
• Can be described by the dihedral angles, called
  f, ?, and ? angles.
• Ramachandran Plot
• Note the crystallographer is not in the
  business of determining molecular composition,
  but determining structural orientation of a
  protein.

4
Introduction toX-Ray Crystallography

• x-rays are used to probe the protein structure
• Why are x-rays used?
• ? Å
• Why are crystals used to do x-ray diffraction?
• Crystals are used because it helps amplify the
  diffraction signal.
• How do the x-rays probe the crystal?
• x-rays interact with the electrons surrounding
  the molecule and reflect. The way they are
  reflected will be prescribed by the orientation
  of the electronic distribution.
• What is really being measured?
• Electron Density!!!

5
Performing X-Ray Crystallography
ExperimentsakaJust Do It

• Braggs Law
• n? 2dsin(?)
• Bragg's Law Applet
• X-Ray Diffraction apparatus.

6
Performing X-Ray Diffraction

• Resultant diffraction pattern from experimental
  setup
• Diffraction pattern is actually a Fourier
  Transform of the electron distribution density.

7
The Fourier Transformand The Inverse Fourier
Transform

• ?

?
8
Are We Finished?

• No!
• 1st We still need to determine the atomic
  construction (all we have is electron
  distribution).
• 2nd There are problems with this analysis
• The phase problem
• Resolution problems
• Solved with Fitting and Refinement

?
9
Structural Basis for Partial Agonist Action at
Ionotropic Glutamate Receptors

• How do partial agonists produce submaximal
  macroscopic currents?
• What is being investigated?
• GluR2 ligand binding core.
• Why is it being investigated?
• Mechanism by which partial agonists produce
  submaximal responses remains to be determined.
• What is going to be done?
• 4 5-R-willardiines will be used as partial
  agonists to determine the structure associated
  with the function.
• Voltage clamping
• X-ray crystallography
• Outside out membrane patches for single channel
  analysis

10
Current Response

• 1st experiment
• Dose Response Analysis using a two-electrode
  voltage clamp on an oocyte expressing the GluR2
  receptor.
• a.) and b.) show affinity of willardiines
• Electronegativity is important
• c.) and d.) show that
• Size does Matter!
• Note relative peak current amplitude with CTZ
• IGlugt IHWgt IFWgt IBrWgt IIW
• Note steady-state current amplitude without CTZ
• IIW gt IBrWgt IFWgt IGlugt IHW
• These data suggests that the efficacy of the XW
  to activate/desensitize the receptor is based on
  size.

11
Structure Meets Function

• Mode of binding appears similar to glutamate
• However, the uracil ring and the X produce a
  crucial structural change in the ligand-binding
  pocket.

12

• Its all about domain closure.
• Hypothesis
• the domains I and II need to be closer to produce
  an opening of ?Pro632
• This opening increases ion conductance.

13
Single Channel Analysis

• They ask the question
• Do receptors populate the same set of
  subconductance states as with full agonists, but
  have different relative frequencies or open
  times?
• To Answer the question, they first performed a
  fluctuation analysis of the macroscopic current
  by
• slowly applying maximally effective
  concentrations of Glu, IW, and HW on outside-out
  membrane patches.
• The weighted average conductance with Glu, HW,
  and IW are 13.1, 11.6, and 7.2 pS.
• Suggests that the reduced efficacy reflects the
  activation of the open states with different
  average conductance.

14
Amplitude and Duration of Open States

• To determine the amplitude and duration of the
  open states, a single channel analysis of the
  steady state responses was carried out.
• Note in a and b, the distributions are the same
  (same conductane), so it must be that the open
  times of the pore for the different ligands are
  different.

15
Towards a Structural View of Gating in Potassium
Channels

• Ion Channel has 3 crucial elements
• Ion conduction pore
• Ion gate
• Voltage sensor
• Architecture of Kv channels
• Channel is a tetramer
• N-terminus of S1 is thought to function as an
  intracellular blocker of the pore, which
  underlies fast inactivationimplies it is inside
  the membrane
• S1-S2 linker glycosylatedoutside of membrane.
• S2-S3 cystein can be modified by MTS.
• S3protein toxins indicate that this is close to
  outside.
• S4 N-terminus is accessible to MTS outside.
• S4 S4-S4 reacts to MTS inside.
• S5-S6 is best defined because it remains well
  conserved across different channels.

16
Gate Structure

• Pore domain is formed by S5 and S6 with S5-S6
  lining the pore.
• KcsA
• x-ray structures support this model.
• QApore blockergets stuck with rapid
  hyperpolarizationgate is on inside.
• Further experiments indicate that the gate is on
  the inside.
• MthK
• Caught in an open state.
• Pore Domains Structure and function
• PVP motif (in many channels)proline tends to
  kink helicies.
• Increased MTS reactivity implies a larger opening
  with the PVP.
• Metal interations not possible in the KcsA or
  MthK models.

17
Voltage Sensors The Competing Models

• S4 region is believed to be the sensor (charge
  rich region)
• S2 S3 have been shown to affect the voltage
  activation relationship.
• Membrane Translocation Model
• Protein charges move large distances through the
  membran.
• Focused Field Model
• Protein charges move smaller distances and focus
  electric field across membrane.

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Model Verification!Or is it?

• Note location of S4
• MT Modelyeah!
• FF Modelawwh!
• Some Problemos
• Possible distortions in x-ray structure of KvAP
• Open and closed structure mixed?
• S1-S2 linkers suppose to be extracellularfrom
  glycosylation sites experiments.
• A number of other problems
• Packing
• MTS reactivity on both sides of membrane with
  approx. the same accessibility, active or not
• Inconsistencies with orientations of other SX
  components in the structure.
• Electron Microscopy shows a more expected
  conformation for the open position
• Most noted discrepancy is that the N-terminus of
  S4 and S3 are probably much closer than what the
  x-ray structure shows.

19
FinallyEvidence for the Models

• MTM
• Fab Fragments show biotin-avidin complexes on
  both sides of the membrane. Voltage sensor
  paddle (S3b-S4)
• Redexternal
• Dark blueinternal
• Yellowboth
• FFM
• Fluorophore attatched to the N-terminal end of S4
  maintains its wavelength
• Energetically more favorable

20
Conclusion

• Presented fundamentals of x-ray crystallography
  and how to interpret the data.
• Presented a paper which discussed structure and
  function using x-ray crystallography with GluR2
  receptors, and
• Discussed another paper that reviewed the current
  accepted structures of Kv receptors and
  problems/inconsistencies with them.