The Bowers’ Groupbowers.chem.ucsb.edu/presentations/asms-may2004/oxytocin.pdf · Zn2+-Oxytocin...

Determination of the Structure of the Zn2+-Oxytocin Complex:

Implications for Oxytocin-Receptor Binding

Alexandra Seuthe, Dengfeng Liu, Oli Th. Ehrler,Xiaohua Zhang, Thomas Wyttenbach and Michael T. Bowers

Oxytocin (OT) StructureCys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)

S SOT

Synthesized in posterior pituitary and released into circulation.

Receptor is G-protein found in smooth muscle cells.

Conformation of OT ligand dramatically affects binding to receptor.

Virtually all vertebrate species have an OT-like hormone:

Disulfide bridge b/t residues 1 and 6Cyclic portion, 3 residue amidated tail

Oxytocin (OT) Function OT is found in equivalent concentration in both sexes.

OT has been linked to several physiological activities:

1) Uterine contractions during birth2) Lactation

Also responsible for Affiliation in mammals.

Establishment of complex social and bonding behaviors related to reproduction and the care of offspring.

i.e: maternal behavior, infant separation distress, mate formation

Metal-OT ComplexesWhy Zinc?

Zinc Fingers: Protein motif that binds DNATranscription Factors necessary for DNA replication.

Essential elements and other metals have been found to form complexes with OT.

i.e.: Cu2+, Zn2+, Co2+, Mg2+, Ca2+, Ni2+

The presence of divalent cation is essential for specific binding of OT to receptor.(Pearlmutter and Soloff, J Biol Chem, 1979)

OT and Divalent Metal Cations

~ 30 residuesOT = 9 residues

Characterize the OT-Zinc complex.Research Objectives

Observation: Question:

OT has high affinity for divalent metal.

What are the binding properties of theOT-Metal complex?

Lock and key model: Receptor binding is conformation-dependent.

Does Zinc cause a conformational change?

Divalent metal cations required for OT-Receptor binding.

Does Zinc cause a conformational change which will enhance bonding?

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)S S

OT

Experimental methods

Mass spectrometry Cross section

(collisions with helium)

Hydration (equilibrium with water vapor)

Theoretical methods

Molecular mechanics Density functional theory

Molecule surface

Molecule structure

Cross section

Cross sectionProtons/Metal ions

Molecule size/shape

Instrumentation

ESI IonSource

ESI IonSource

IonFunnel

IonFunnel

DriftCell

DriftCell MSMS DetectorDetector

Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer

DetectorTo PumpTo PumpTo Pump

To Pump

Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Instrumentation

ESI IonSourceESI IonSource

IonFunnel

IonFunnel

DriftCell


Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Instrumentation

ESI IonSource

ESI IonSource

IonFunnel

IonFunnel

DriftCell


Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Instrumentation

IonFunnel

IonFunnel

DriftCell


ESI IonSource

ESI IonSource

Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Instrumentation

ESI IonSource

ESI IonSource

IonFunnel

IonFunnel

DriftCell


Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Ion Funnel

DriftCell

Ion Optics

QuadAnalyzer


To Pump

Cross section measurements

ESI IonSource

ESI IonSource

IonFunnel

IonFunnel

DriftCell

DriftCell MSMS DetectorDetectorDriftCell

DriftCell

Hydration under equilibrium conditions

ESI IonSource

ESI IonSource

IonFunnel

IonFunnel

DriftCell

DriftCell MSMS DetectorDetectorDriftCell

DriftCell

in out

E

Drift cell12 torr H2O

M+ M+(H2O)n

ratio ofpeak intensities

equilibriumconstant

vant Hoff

∆H° and ∆S°

Data Analysis

[O T + H ]+

[O T + H + K ]2 +

[O T + H + N a ]2 +

[O T + 2 H ]2 ++2

[OT + H]++1

Experimental ResultsMassSpectrumOxytocin

m/z2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0

[OT] = 50 µM

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0

[O T + H ]+

[O T + H + K ]2 +

[O T + H + N a ]2 +

[O T + 2 H ]2 +

m/z

[OT + 2 H]2+

[OT + H]+

[O T + H

[OT + H + K]2+


H3N+−

[OT + H + Na]2+MassSpectrumOxytocin

[OT] = 50 µM

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0

[O T + H ]+

[O T + H + K ]2 +

[O T + H + N a ]2 +

[O T + 2 H ]2 +

m/z

[OT + 2 H]2+

[OT + H]+

[O T + H

[OT + H + K]2+


H3N+−

H+, Na+, K++

[OT + H + Na]2+MassSpectrumOxytocin

[OT] = 50 µM

Theory Results: OT-H+Bare OT

Theory: MM (Simulated Annealing) - CHARMM force fieldDFT - SVP basis set; BP86 functional

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)S SOT

228 Å2 (calc)230 Å2 (exp)

Hydration data

(OT+H)+ value is consistent with acompletely buried ammonium group.

(OT+H)+7(OT+H)+

7Buried NH3+

15Exposed NH3+

Water binding energy (kcal/mol)

[1]

[1]

[1] Lui et al. JACS 2003, 125, 8458

m/z2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0

[O T + H ]+

[O T + H + K ]2 +

[O T + H + N a ]2 +

[O T + 2 H ]2 +

[OT + H]+[OT + H]+

[OT + 2 H]2+

MassSpectrumOxytocin

[OT] = 50 µM

[O T + H ]+

[O T + H + K ]2 +

[O T + H + N a ]2 +

[O T + 2 H ]2 +

m/z

with ZnCl2

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0

[O T + Z n ]2 ++ Z n C l2

n o (O T +2 H )2 + !!

O T + Z n C l2

[O T + Z n ]2 +

[OT + H]+

[OT + Zn]2+

[OT + 2 H]2+

[Zn2+] = 200 µM[OT] = 50 µM


[O T + H ]+

[O T + H + K ]2 +

[O T + H + N a ]2 +

[O T + 2 H ]2 +

m/z

with ZnCl2

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0

[O T + Z n ]2 ++ Z n C l2

n o (O T +2 H )2 + !!

O T + Z n C l2

[O T + Z n ]2 +

[OT + H]+

[OT + 2 H]2+

[OT + Zn]2+

[Zn2+] = 200 µM[OT] = 50 µM


Theory Results: OT-Zn2+ ComplexTheory: MM (Simulated Annealing) - CHARMM force field

DFT - SVP basis set; BP86 functional

Zn2+

ZnO distance = 204-215 pmZn2+ + O ionic radaii = 214 pm

236 Å2 (calc)236 Å2 (exp)


octahedralcoordination

sphere

donating backbone carbonyl oxygen

Hydration data

29Zn2+(H2O)5

10(OT+Zn)2+

10(Peptide)2+

96Bare Zn2+

Water binding energy (kcal/mol)

(OT+Zn)2+ [1]

Zn2+

[1]

[2]

(OT+Zn)2+ value is consistent with a completely buried zinc ion.

Water interacts with peptide surface.[1] Bock et al. JACS 1995, 117, 3754 [2] Lui et al. JACS 2003, 125, 8458

Summary of structural differencesbetween bare OT and OT-zinc complex

bare OT OT-zinc complex

Zn2+

Zn2+ buried helical backbone C-terminus distant

from N-terminus

N-terminus buried N- and C-termini close

Are these structural differences relevant for OT-receptor binding?

Oxytocin (OT) Receptor Interaction

OT receptor sequence is known, but ligand binding is not.

The cyclic portion of OT binds to extracellular loop.The linear portion binds to another extracellular loop.

Oxytocin (OT) Receptor InteractionWhy do divalent metals increase OT binding?


Residue 3-5 are found to be crucial for cyclic hormone-receptor interaction.

Hydrophobic residue Ile/Phe-3 forms hydrophobic pocket interaction

Conserved Gln-4 and Asn-5 interact with conserved residues in the receptor.

Side Chain Conformation

Ile3Gln4

Asn5Ile3Gln

4

Asn5

Residues 3-5 are found to be crucial for cyclic hormone-receptor interaction.Zinc lines up side chains of residues 3-5.

OT + Zn2+bare OT

N-terminus Conformation

OT + Zn2+

N-terminusexposed

Interaction of the cyclic portion is stabilized by salt bridge interaction between N-terminus and GlutamicAcid in receptor.

N-terminusburied

Zinc displaces N-terminus from

the interior of peptide.

bare OT

Compact vs. Extended

! Bare OT! OT- Zinc Complex

Isotocin (IT)

Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly(NH2)S S

IT


Osteichthyes Bony Fish

Isotocin (IT) (Isotocin+Zn)2+

Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly(NH2)S S

IT


Osteichthyes Bony Fish

donating backbone oxygen

IT OT

ConclusionsQuestion:Observation:


What are the binding properties of the OT-metal complex?

Experiments confirm that Zinc is buried in peptide structure.






Yes.






Yes.

Divalent metal cations required for OT-Receptor binding.

Does Zinc cause a conformational change which will enhance bonding?

ConclusionsWhen zinc binds to ligand, a dramatic conformation changeoccurs which appears more favorable for binding.

Acknowledgements

Dengfeng LiuOli Th. EhrlerXiaohua Zhang

Professor Bowers

Bowers GroupDr. Thomas WyttenbachDr. Catherine Carpenter

Determination of the Structure of the Zn2+-Oxytocin Complex: Implications for Oxytocin-Receptor BindingExperimental methodsInstrumentationExperimental ResultsTheory Results: OT-Zn2+ ComplexHydration dataSummary of structural differencesbetween bare OT and OT-zinc complexOxytocin (OT) Receptor InteractionOxytocin (OT) Receptor InteractionCompact vs. ExtendedIsotocin (IT)Isotocin (IT)

The Bowers’ Groupbowers.chem.ucsb.edu/presentations/asms-may2004/oxytocin.pdf · Zn2+-Oxytocin...

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