X-Ray CrystallographyX-Ray Crystallographyandand

It’s ApplicationsIt’s Applications

ByByBernard FendlerBernard Fendler

andandBrad GrovemanBrad Groveman

IntroductionIntroduction

Present basic concepts of protein structurePresent basic concepts of protein structure Discuss why x-ray crystallography is used to Discuss why x-ray crystallography is used to

determine protein structuredetermine protein structure Lead through x-ray diffraction experimentsLead through x-ray diffraction experiments And present how to utilize experimental And present how to utilize experimental

information to design structural models of information to design structural models of proteinsproteins

Introduction to Protein Structure: Introduction to Protein Structure: ““The Crystallographer’s Problem”The Crystallographer’s Problem”

What is the crystallographer’s What is the crystallographer’s problem?: Structural problem?: Structural Determination!Determination!

Structure ~ FunctionStructure ~ Function Amino acids are strung together Amino acids are strung together

on a carbon chain backbone.on a carbon chain backbone. As a result:As a result:

Can be described by the dihedral Can be described by the dihedral angles, called angles, called φφ, , ψψ, and , and ωω angles. angles.

Ramachandran PlotRamachandran Plot Note: the crystallographer is not Note: the crystallographer is not

in the business of determining in the business of determining molecular composition, but molecular composition, but determining structural orientation determining structural orientation of a protein.of a protein.

Introduction to:Introduction to:X-Ray CrystallographyX-Ray Crystallography

x-rays are used to probe the x-rays are used to probe the protein structure:protein structure:

Why are x-rays used?Why are x-rays used? λλ ~ Å ~ Å

Why are crystals used to do x-ray Why are crystals used to do x-ray diffraction?diffraction? Crystals are used because it helps Crystals are used because it helps

amplify the diffraction signal.amplify the diffraction signal. How do the x-rays probe the How do the x-rays probe the

crystal?crystal? x-rays interact with the electrons x-rays interact with the electrons

surrounding the molecule and surrounding the molecule and “reflect”. The way they are “reflect”. The way they are reflected will be prescribed by the reflected will be prescribed by the orientation of the electronic orientation of the electronic distribution.distribution.

What is really being measured?What is really being measured? Electron Density!!!Electron Density!!!

Performing X-Ray Crystallography ExperimentsPerforming X-Ray Crystallography Experimentsakaaka

“Just Do It”“Just Do It”

Bragg’s Law:Bragg’s Law: nnλλ =2d =2dsin(sin(θθ)) Bragg's Law AppletBragg's Law Applet

X-Ray Diffraction X-Ray Diffraction apparatus.apparatus.

Performing X-Ray DiffractionPerforming X-Ray Diffraction

Resultant diffraction Resultant diffraction pattern from pattern from experimental setupexperimental setup

Diffraction pattern is Diffraction pattern is actually a Fourier actually a Fourier Transform of the Transform of the electron distribution electron distribution density.density.

The Fourier TransformThe Fourier Transformand and

The Inverse Fourier TransformThe Inverse Fourier Transform

Are We Finished?Are We Finished? No! No!

11stst: We still need to determine the atomic construction (all we : We still need to determine the atomic construction (all we have is electron distribution).have is electron distribution).

22ndnd: There are problems with this analysis:: There are problems with this analysis: The phase problemThe phase problem Resolution problemsResolution problems

Solved with Fitting and RefinementSolved with Fitting and Refinement

Structural Basis for Partial Agonist Action at Structural Basis for Partial Agonist Action at Ionotropic Glutamate ReceptorsIonotropic Glutamate Receptors

How do partial agonists produce How do partial agonists produce submaximal macroscopic submaximal macroscopic currents?currents?

What is being investigated?What is being investigated? GluR2 ligand binding core.GluR2 ligand binding core.

Why is it being investigated?Why is it being investigated? Mechanism by which partial Mechanism by which partial

agonists produce submaximal agonists produce submaximal responses remains to be responses remains to be determined.determined.

What is going to be done?What is going to be done? 4 5-‘R’-willardiines will be used as 4 5-‘R’-willardiines will be used as

partial agonists to determine the partial agonists to determine the structure associated with the structure associated with the function.function.

Voltage clampingVoltage clamping X-ray crystallographyX-ray crystallography Outside out membrane patches Outside out membrane patches

for single channel analysisfor single channel analysis

Current ResponseCurrent Response 11stst experiment: experiment:

Dose Response Analysis using a Dose Response Analysis using a two-electrode voltage clamp on an two-electrode voltage clamp on an oocyte expressing the GluR2 oocyte expressing the GluR2 receptor.receptor.

a.) and b.) show affinity of a.) and b.) show affinity of willardiineswillardiines

Electronegativity is importantElectronegativity is important c.) and d.) show that: c.) and d.) show that:

Size does Matter!Size does Matter! Note relative peak current Note relative peak current

amplitude with CTZ:amplitude with CTZ: IIGluGlu> I> IHWHW> I> IFWFW> I> IBrWBrW> I> IIWIW

Note steady-state current Note steady-state current amplitude without CTZ:amplitude without CTZ:

IIIWIW > I > IBrWBrW> I> IFWFW> I> IGluGlu> I> IHWHW

These data suggests that the These data suggests that the efficacy of the XW to efficacy of the XW to activate/desensitize the receptor activate/desensitize the receptor is based on size.is based on size.

Structure Meets FunctionStructure Meets Function

Mode of binding Mode of binding appears similar to appears similar to glutamateglutamate

However, the uracil However, the uracil ring and the X ring and the X produce a crucial produce a crucial structural change in structural change in the ligand-binding the ligand-binding pocket.pocket.

Its all about domain Its all about domain closure.closure. Hypothesis:Hypothesis:

the domains I and II the domains I and II need to be closer to need to be closer to produce an opening of produce an opening of ΔΔPro632 Pro632

This opening increases This opening increases ion conductance.ion conductance.

Single Channel AnalysisSingle Channel Analysis They ask the question:They ask the question:

Do receptors populate the same Do receptors populate the same set of subconductance states as set of subconductance states as with full agonists, but have with full agonists, but have different relative frequencies or different relative frequencies or open times?open times?

To Answer the question, they first To Answer the question, they first performed a fluctuation analysis of performed a fluctuation analysis of the macroscopic current by the macroscopic current by slowly applying maximally slowly applying maximally

effective concentrations of Glu, effective concentrations of Glu, IW, and HW on outside-out IW, and HW on outside-out membrane patches.membrane patches.

The weighted average The weighted average conductance with Glu, HW, and conductance with Glu, HW, and IW are 13.1, 11.6, and 7.2 pS.IW are 13.1, 11.6, and 7.2 pS.

Suggests that the reduced Suggests that the reduced efficacy reflects the activation of efficacy reflects the activation of the open states with different the open states with different average conductance.average conductance.

Amplitude and Duration of Open Amplitude and Duration of Open StatesStates

To determine the To determine the amplitude and duration of amplitude and duration of the open states, a single the open states, a single channel analysis of the channel analysis of the steady state responses steady state responses was carried out.was carried out.

Note in a and b, the Note in a and b, the distributions are the same distributions are the same (same conductane), so it (same conductane), so it must be that the open must be that the open times of the pore for the times of the pore for the different ligands are different ligands are different.different.

Towards a Structural View of Towards a Structural View of Gating in Potassium ChannelsGating in Potassium Channels

Ion Channel has 3 crucial elements:Ion Channel has 3 crucial elements: Ion conduction poreIon conduction pore Ion gateIon gate Voltage sensorVoltage sensor

Architecture of KArchitecture of Kvv channels channels Channel is a tetramerChannel is a tetramer N-terminus of S1 is thought to function N-terminus of S1 is thought to function

as an intracellular blocker of the pore, as an intracellular blocker of the pore, which underlies fast inactivation—which underlies fast inactivation—implies it is inside the membraneimplies it is inside the membrane

S1-S2 linker glycosylated—outside of S1-S2 linker glycosylated—outside of membrane.membrane.

S2-S3 cystein can be modified by S2-S3 cystein can be modified by MTS.MTS.

S3—protein toxins indicate that this is S3—protein toxins indicate that this is close to outside.close to outside.

S4 N-terminus is accessible to MTS S4 N-terminus is accessible to MTS outside.outside.

S4 & S4-S4 reacts to MTS inside.S4 & S4-S4 reacts to MTS inside. S5-S6 is best defined because it S5-S6 is best defined because it

remains well conserved across remains well conserved across different channels.different channels.

Gate StructureGate Structure Pore domain is formed by S5 and Pore domain is formed by S5 and

S6 with S5-S6 lining the pore.S6 with S5-S6 lining the pore. KcsAKcsA

x-ray structures support this x-ray structures support this model.model.

QA—pore blocker—gets stuck QA—pore blocker—gets stuck with rapid hyperpolarization—gate with rapid hyperpolarization—gate is on inside.is on inside.

Further experiments indicate that Further experiments indicate that the gate is on the inside.the gate is on the inside.

MthKMthK Caught in an open state.Caught in an open state.

Pore Domains Structure and Pore Domains Structure and functionfunction PVP motif (in many channels)—PVP motif (in many channels)—

proline tends to kink helicies.proline tends to kink helicies. Increased MTS reactivity implies a Increased MTS reactivity implies a

larger opening with the PVP.larger opening with the PVP. Metal interations not possible in Metal interations not possible in

the KcsA or MthK models.the KcsA or MthK models.

Voltage Sensors: Voltage Sensors: The The

Competing ModelsCompeting Models S4 region is believed to be the S4 region is believed to be the

sensor (charge rich region)sensor (charge rich region) S2 & S3 have been shown to S2 & S3 have been shown to

affect the voltage activation affect the voltage activation relationship.relationship.

Membrane Translocation Membrane Translocation ModelModel Protein charges move large Protein charges move large

distances through the distances through the membran.membran.

Focused Field ModelFocused Field Model Protein charges move smaller Protein charges move smaller

distances and focus electric distances and focus electric field across membrane.field across membrane.

Model Verification!Model Verification!Or is it?Or is it?

Note location of S4Note location of S4 MT Model=yeah!MT Model=yeah! FF Model=awwh!FF Model=awwh!

Some ProblemosSome Problemos Possible distortions in x-ray Possible distortions in x-ray

structure of KvAPstructure of KvAP Open and closed structure mixed?Open and closed structure mixed? S1-S2 linkers suppose to be S1-S2 linkers suppose to be

extracellular—from glycosylation extracellular—from glycosylation sites experiments.sites experiments.

A number of other problemsA number of other problems PackingPacking MTS reactivity on both sides of MTS reactivity on both sides of

membrane with approx. the same membrane with approx. the same accessibility, active or notaccessibility, active or not

Inconsistencies with orientations of Inconsistencies with orientations of other SX components in the other SX components in the structure.structure.

Electron Microscopy shows a more Electron Microscopy shows a more expected conformation for the open expected conformation for the open positionposition

Most noted discrepancy is that the Most noted discrepancy is that the N-terminus of S4 and S3 are N-terminus of S4 and S3 are probably much closer than what the probably much closer than what the x-ray structure shows.x-ray structure shows.

Finally:Finally:Evidence for the ModelsEvidence for the Models

MTM: MTM: Fab Fragments show Fab Fragments show

biotin-avidin complexes on biotin-avidin complexes on both sides of the both sides of the membrane. Voltage membrane. Voltage sensor paddle (S3b-S4)sensor paddle (S3b-S4) Red=externalRed=external Dark blue=internalDark blue=internal Yellow=bothYellow=both

FFM:FFM: Fluorophore attatched to Fluorophore attatched to

the N-terminal end of S4 the N-terminal end of S4 maintains its wavelengthmaintains its wavelength

Energetically more Energetically more favorablefavorable

ConclusionConclusion

Presented fundamentals of x-ray crystallography Presented fundamentals of x-ray crystallography and how to interpret the data.and how to interpret the data.

Presented a paper which discussed structure Presented a paper which discussed structure and function using x-ray crystallography with and function using x-ray crystallography with GluR2 receptors, andGluR2 receptors, and

Discussed another paper that reviewed the Discussed another paper that reviewed the current accepted structures of Kcurrent accepted structures of Kvv receptors and receptors and

problems/inconsistencies with them.problems/inconsistencies with them.