National Institutes of HealthNational Science FoundationFlorida State University Research FoundationState of Florida
Department of Chemistry & BiochemistryInstitute of Molecular Biophysics
National High Magnetic Field LaboratoryFlorida State University
Tallahassee, Florida
Charcterization of the M2 Proton Channel
Functional Mechanism from Influenza A
Obtained from Lipid Bilayer Preparations
Winter School January 2016
Neuraminidase
Hemaglutinin
M2
The “Spanish Flu” Pandemic of 1918-1919
Makeshift hospitals in the US
• 1/3 of the world’s population was infected
• 3% of the world’s population died (50 to 100 million people)
• In the US, 28% of the population was infected
• In the US, 500,000 to 650,000 died
• Most fatalities were healthy young adults
Brief Outline
• Intro to M2 and Influenza
• Intro to Oriented Sample solid state NMR
• M2 structure Determination in Lipid Bilayers
• Structural Comparisons
• Magic Angle Spinning solid state NMR Results
• The role of HxxxW in Proton Conductance
2
Influenza A Viral Life Cycle
M2 ProtonChannel
Adapted from Lamb & Krug, 19963
Influenza A Viral Life Cycle
Rossman & Lamb, 2011
Ç1 2 3
321
4
M S L LT
EVET
P I R N E WG CR
CNDSSDPL
VV
A AS
II G
IL
HL
IL W
IL
DR
L
N
E
T
S A
FF
KC
I
YR
FF
E
H
GL
K
RG P T GE
E
S
E
AV
S D
PV
Y R M S
E
K R
SQ QE D AD
EL
E I S HVF
M2 Proton
Channel
Amino Acid
Sequence
Amino Terminus: 2 Cys
residues form crosslinks
Amphipathic Helix that
binds lipid membrane
surface
Carboxy-
Terminus:
M1 Binding
Domain
Transmembrane Helix
essential for proton
conductance
Viral
Membrane
Aqueous Viral Interior
Aqueous Viral Exterior
0
40
80
120
160
6.5 6 5.5
H+/
tetr
amer
/s
pHex
kDa
50
252015
10
M2(22-62)
• Liposomal Assays show H+ conductance
similar to full length protein & similar
sensitivity to amantadine.
• Similar to Schnell & Chou, 2008 The
conductance domain is shown to be a tetramer
running at a slightly higher molecular weight
that the predicted 19 kDa.
SDS PAGE
6.5 6.0 5.5pH
40
80
120
160
H+ /
Tetr
ame
r/s
0
500
1000
2000
1500
H+ /
Tetr
ame
r
0-5 5 10 15 20Time (s)
M2 Conductance Domain
w/o AMT
w AMT
w/o AMT
w AMT
0
Emily Peterson and David Busath, BYUPacifichem - Honolulu - 12/16/2010
Some of the Data for the M2 Conductance Domain Proton Channel Structure
• A tetrameric structure that shows some dimer of dimer character, but only subtle evidence in the OS ssNMRspectra.
Viral Exterior
M2 Proton Channel Characterized by ssNMR in DOPC/DOPG Lipids
The structure restrained primarily by orientational restraints and refined using restrained MD all in the same lipid bilayer
The structure of the H37 tetrad and Trp41 tetrad were refined using QM/MM calculations
V27
H37
W41
Sharma et al., 2010 Science
Viral External
Pore
Viral
Internal Pore
Viral Interior
Viral Interior
Viral Exterior
His-His+ Pair
Scarce Water in pore
due to Trp41 residues
In the pore – the primary
gate for proton
conductance
Abundant Water in pore
due to Gly 34 residues
In the pore
Waters & Lipids Associated with the M2 Conductance
Domain Structure
His37 Labeled Full–Length M2 (H57,90Y)
pH 7.3 pH 6.6
Figure 5.1 Aromatic regions of 2D 13
C-13
C correlation spectra with 50ms mixing
time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)
pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold
symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch
H37) is
labeled in (C); The chemical exchange peaks are labeled in blue and the
short-distance correlations are labeled in green in (B); The additional neutral
conformation t5 is labeled in (B).
a
b
Figure 5.1 Aromatic regions of 2D 13
C-13
C correlation spectra with 50ms mixing
time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)
pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold
symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch
H37) is
labeled in (C); The chemical exchange peaks are labeled in blue and the
short-distance correlations are labeled in green in (B); The additional neutral
conformation t5 is labeled in (B).
a
b
13C (ppm)
13C
(p
pm
)1
3C
(p
pm
)
Miao et al., 2015 Structure
50 ms DARR spectra
@ -10°C In DOPC/DOPE
as in PDB: 2L0J
>>Dimer of Dimer? >>Additional Complexity
3B
KD
Sto
uff
er e
t al
., 2
00
813C-13C Correlation Spectra of His37 at lower pH
pH 6.6 pH 6.2 pH 5.8
Figure 5.1 Aromatic regions of 2D 13
C-13
C correlation spectra with 50ms mixing
time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)
pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold
symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch
H37) is
labeled in (C); The chemical exchange peaks are labeled in blue and the
short-distance correlations are labeled in green in (B); The additional neutral
conformation t5 is labeled in (B).
a
b
13C
(p
pm
)
pH 6.6 pH 6.2 pH 5.8
13C (ppm)
Miao et al., Structure, 2015
Figure 5.1 Aromatic regions of 2D 13
C-13
C correlation spectra with 50ms mixing
time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)
pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold
symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch
H37) is
labeled in (C); The chemical exchange peaks are labeled in blue and the
short-distance correlations are labeled in green in (B); The additional neutral
conformation t5 is labeled in (B).
a
b
50 ms DARR spectra
@ -10°C
Even More Complexity
3B
KD
Sto
uff
er e
t al
., 2
00
8
200220240
15N (ppm)
180 160260
His37 pH Titration of Full Length M2 in DOPC/DOPE Bilayers.
Miao et al., Structure, 2015
Charge
pH7.0 6.5 6.0 5.5 5.0 4.57.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
pKas: 6.3 ± 0.16.3 ± 0.1
5.5 ± 0.3
Broadened Lines Dynamics & Heterogeneity
>>Cooperative Protonation of His37
3B
KD
Sto
uff
er e
t al
., 2
00
815N Spin Echo Spectra of pH 6.2 spectra
Miao et al., Structure, 2015
0.167 ms
5.0 ms
2.5 ms
Heterogeneous broadening
3B
KD
Sto
uff
er e
t al
., 2
00
8Both p & t uncharged states observed at high pH
13C (ppm)
15N
(pp
m)
t1t2
t3
p1
Figure 5.1 Aromatic regions of 2D 13
C-13
C correlation spectra with 50ms mixing
time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)
pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold
symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch
H37) is
labeled in (C); The chemical exchange peaks are labeled in blue and the
short-distance correlations are labeled in green in (B); The additional neutral
conformation t5 is labeled in (B).
a a
bb
Cg/Nd1Ce1/Nd1
Cd2/Ne2Ce1/Ne2
Cd2/Ne2Ce1/Ne2
Ce1/Nd1
Cg/Nd1
Miao et al., Structure, 2015
NC zf TEDOR Spectra
1 ms mixing time
pH 7.3 & -10°C Full Length M2
3B
KD
Sto
uff
er e
t al
., 2
00
8NC Spectra of the His37 at pH 7.3 & 6.2
13C (ppm)
pH 7.3
pH 6.6
Figure 5.1 Aromatic regions of 2D 13
C-13
C correlation spectra with 50ms mixing
time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)
pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold
symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch
H37) is
labeled in (C); The chemical exchange peaks are labeled in blue and the
short-distance correlations are labeled in green in (B); The additional neutral
conformation t5 is labeled in (B).
15N (ppm)
Miao et al., Structure, 2015
195238253 170- - - -
255
170
160
180
190
200
170
160
180
260
250
160
260
250
160
170
180
15N
(p
pm
)
245
235
190
180
170
160
pH 6.2
Stable Structural Heterogeneity
Figure 5.1 Aromatic regions of 2D 13
C-13
C correlation spectra with 50ms mixing
time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)
pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold
symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch
H37) is
labeled in (C); The chemical exchange peaks are labeled in blue and the
short-distance correlations are labeled in green in (B); The additional neutral
conformation t5 is labeled in (B).
N••••H––N
M2FL NC Spectra of the His37 at pH 6.2
NP
P
NP
P
PP
• Imidazole-Imidazolium
H-bonded pairs
15N
(p
pm
)
13C (ppm)
170
160
180
190
235
245
255
170
160
180
190
200
t +
Miao et al., Structure, 2015
His Tetrad Chemistry Driven by Charge Delocalization, Structural Stability
Separated Charges:
No H-Bond
Solvated His-His+
Dong et al., Chem. Sci. RSC, 2013
His-His+ Short H-bonds Confirmed in Full Length M2
15N (ppm)
1H (ppm)
5
10
15
20
160200 180
••••
••••
Miao et al., Structure, 2015
+
+t
His+ protons exchange with water
His-His+ H-bonds associated with
high 1H and 15N frequenciesx
HN HETCOR
500 µs contact time
pH 6.2
-10°C
Multiple His-His+ States
His-His+ states exchange with
H2O-His+ states
193 ppm
188 ppm
3B
KD
Sto
uff
er e
t al
., 2
00
8Some Limits on the Exchange Rates
pH 6.2
15N (ppm)
1H (ppm)
5
10
15
20
160200 180
••
••
3600 Hz
4800 Hz
300 Hz
In 1H dimension:
4800 Hz > Exchange Rate > 0.2 Hz
Hydronium attack on the neutral His
In 15N dimension
300 Hz > Exchange Rate
3B
KD
Sto
uff
er e
t al
., 2
00
8Evidence for Short H-Bonds near Physiological Temperature
15N (ppm)
1H (ppm)
The stability of the multiple states suggests:• these states are not dictated by sidechains• not by the very stable backbone structure• but by the oligomeric helix packing
5
10
15
20
160200 180
••
••
Miao et al., Structure, 2015
At 23°C 15N frequencies associated with short H-bonds are present
At 23°C the His-His+ crosspeaks are not directly observed, but must be present since
resonces at 190 ppm are observed
Ch1 Ce1 – t1 Ce1
Ch1 Ce1 - t2 Ce1Ch1 Cg - t2 Cg
Ch1 Cd2 - t2 Ce1
Ch1 Ce1 – t1 Cd2
13C ppm
13C ppm
Evidence for both Exchange and Distances in the His37 Tetrad
Here exchange and
distances from just one
of the charged state
(Ch1) are identified.
Miao et al., 2015 Structure
pH 5.8 50 ms DARR
Sidechain Structural Dynamics at the TM – TM Interface
An Explanation for His-His+ Heterogeneity
3B
KD
Sto
uff
er e
t al
., 2
00
8HN Spectra of the His37 at pH 6.2 at -10°C (red) & 23°C (blue) – M2
15N (ppm)
1H
(ppm
)
5
10
15
20
160200 180
••
••
Miao et al., 2015 Structure
Viral Interior
Viral Exterior
His-His+ Pair
Scarce Water in pore
due to Trp41 residues
In the pore – the primary
gate for proton
conductance
Abundant Water in pore
due to Gly 34 residues
In the pore
Waters & Lipids Associated with the M2 Conductance Domain
Structure
Viral Exterior
Viral Interior
Attack by hydronium appears to
be sub-msec timescale
Conductance is known to be ~100/s
Maybe even less frequent at -10°C
A Futile Cycle in which hydronium attacks
from the external pore and the proton is
reabsorbed by a water in the aqueous pore
---
Evidence for a Futile Cycle
M2 Conductance Mechanism • Hydronium ion attack of the +2 state resulting
in a +3 state and breaking a short H-bond.
• A proton can be absorbed by
water from either the external or
internal pore
• If the proton is absorbed
into the external pore
a Futile Cycle results
• If the proton is absorbed
into internal pore a
Conductance Cycle results
Miao et al., Structure, 2015
Cholesterol Stabilizes the
Amphipathic Helix
Mixed sample of:
13C Leucine FLM2 and
13C Phenylalanine FLM2
Slices through Leu Ca
w/o AMT & w/o Cho
w AMT & w/o Cho
w AMT & w Cho
Ekanayake et al., in press, BJ
Distances in Red represent distances
From the 2L0J M2 structure
Slice through C25
Slice through C3
13CLabeled Phenylalanine FLM2
& Labeled Cholesterol
Cholesterol and M2FL
Phe Cg
Phe Cd,e
Ekanayake et al., in press, BJ
A Typical Cholesterol binding
pocket with:
• C3 close to Phe 54
• Cholesterol in Helix-Helix Crevice
• Cholesterol close to palmitylation
site, Cys50.
Fourth & Final Set of Conclusions:
• M2 has a unique Histidine Tetrad that is a His–His+ Dimer of Dimers Structure
• M2 Full Length Protein Displays Exchange and Dynamic Processes to Facilitate Proton Exchange
• M2 Conductance is a Combination of a Futile and Conductance Cycles
• Biological Chemistry Requires a Molecular Framework on which to Hang the Chemistry & Dynamics to Facilitate the Chemistry
• Solid State NMR can Uniquely Characterize the Structural and Dynamic Details in Membrane Proteins in a variety of model environments and in cell membranes
Just Some of those
who contributed so
much to my lab