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
DetectorTo PumpTo PumpTo Pump
To Pump
Instrumentation
ESI IonSourceESI 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
DetectorTo PumpTo PumpTo Pump
To Pump
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
DetectorTo PumpTo PumpTo Pump
To Pump
Instrumentation
IonFunnel
IonFunnel
DriftCell
DriftCell MSMS DetectorDetector
ESI IonSource
ESI IonSource
Ion Funnel
DriftCell
Ion Optics
QuadAnalyzer
DetectorTo PumpTo PumpTo Pump
To Pump
Ion Funnel
DriftCell
Ion Optics
QuadAnalyzer
DetectorTo PumpTo PumpTo Pump
To Pump
Instrumentation
IonFunnel
IonFunnel
DriftCell
DriftCell MSMS DetectorDetector
ESI IonSource
ESI IonSource
Ion Funnel
DriftCell
Ion Optics
QuadAnalyzer
DetectorTo PumpTo PumpTo Pump
To Pump
Ion Funnel
DriftCell
Ion Optics
QuadAnalyzer
DetectorTo PumpTo PumpTo Pump
To Pump
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
DetectorTo PumpTo PumpTo Pump
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+
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)S S
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+
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)S S
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
MassSpectrumOxytocin
[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
MassSpectrumOxytocin
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)
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)S SOT
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?
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)S SOT
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
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)S SOT
Osteichthyes Bony Fish
Isotocin (IT) (Isotocin+Zn)2+
Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly(NH2)S S
IT
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly(NH2)S SOT
Osteichthyes Bony Fish
donating backbone oxygen
IT OT
ConclusionsQuestion:Observation:
OT has high affinity for divalent metal.
What are the binding properties of the OT-metal complex?
Experiments confirm that Zinc is buried in peptide structure.
ConclusionsQuestion:Observation:
OT has high affinity for divalent metal.
What are the binding properties of the OT-metal complex?
Lock and key model: Receptor binding is conformation-dependent.
Does Zinc cause a conformational change?
Yes.
ConclusionsQuestion:Observation:
OT has high affinity for divalent metal.
What are the binding properties of the OT-metal complex?
Lock and key model: Receptor binding is conformation-dependent.
Does Zinc cause a conformational change?
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)