New and improved insulin: ^1H{^(19)F} NOE NMR difference spectra for CF_3-substituted aromatic carboxylates bound at the HisB10 sites of the R_6 human insulin (HI) hexamer show strong NOEs between the CF_3 groups and the LeuB6, AsnB3, and PheB1 sidechains. The NOEs and structural modeling establish that these carboxylates form closed complexes with the HisB10 site capped by the PheB1 rings

Bonaccio, Maria

Borchardt, Dan

Dunn, Michael F.

Ghaderi, Nima

Keidel, Donald

Niks, Dimitri

English

Caltech Authors - Main

DOI: 10.1002/cbic.200800746
1HACHTUNGTRENNUNG{19F} NOE NMR Structural Signatures of the Insulin R6 Hexamer: Evidence
of a Capped HisB10 Site in Aryl- and Arylacryloyl-carboxylate Complexes
Donald Keidel,[b] Maria Bonaccio,[a] Nima Ghaderi,[c] Dimitri Niks,[a] Dan Borchardt,[d] and Michael F. Dunn*[a]
Although transplantation and stem cell therapies look promis-
ing, at present, type 1 (insulin-dependent) diabetics can be
treated only with insulin or insulin analogues.[1, 2] Hexameric in-
sulin is widely used in pharmaceutical formulations for treat-
ment of insulin-dependent diabetes mellitus.[3, 4] The stability
and dynamic properties of human insulin (HI) zinc hexamer for-
mulations are critically influenced by allosteric effectors.[3–17]
Therefore, the discovery of ligands with enhanced allosteric
and/or pharmacological properties is important for the design
of improved formulations.[3, 4]
Insulin hexamers exhibit positive and negative cooperativity
and half-of-the-sites reactivity in ligand binding.[3–17] The HI
hexamer undergoes allosteric transitions among three well-
characterized protein conformations, which are designated T6,
T3R3, and R6.
[5–17] Crystalline and precipitated T3R3 and R6 hex-
amers are formulated as slow-release forms,[3, 4] and are stabi-
lized by the binding of allosteric
ligands at two loci, the “phenolic
pockets” (3 in T3R3 and 6 in R6),
and the “HisB10 zinc sites” (1 in
T3R3 and, 2 in R6).
[3, 4, 13–16] The R-
state HisB10 sites (Figure 1) bind
monovalent anions (halides,
pseudo halides, and carboxy-
lates).[3, 4, 13–16] Binding interac-
tions are structurally well charac-
terized at the phenolic pockets[9–12]
but not at the HisB10 sites. Each
HisB10 site is formed by a three-
helix bundle consisting of B-
chain residues 1–9 situated
around the hexamer three-fold
symmetry axis, to create 12 
deep amphipathic cavities.[4, 9–12]
These cavities extend from the protein surface to the HisB10
ZnII (Figure 1). Monoanions bind in these cavities and coordi-
nate to ZnII and give pseudotetrahedral ZnII–(His)3-X
 com-
plexes (X= Cl or SCN),[9–16, 18–21] (Figure 1) or 5-coordinate
complexes with carboxylates.[12–14, 19–21] This 1H ACHTUNGTRENNUNG{19F} NOE study
was undertaken to identify weak bonding interactions be-
tween the ligand and the cavity residues that stabilize the
binding of organic carboxylates to the HisB10 zinc sites.
The binding of carboxylates to the HisB10 site has been ex-
tensively investigated in solution, and it is unambiguously es-
tablished that binding involves coordination to the HisB10 zinc
ions; [6, 8, 16, 18–21] however, no X-ray structures of R-state carbox-
ylate complexes have been published. Aromatic carboxylates
make contacts with the HisB10 cavity walls that contribute
substantially to the binding free energy.[3, 8, 19–21] The T3R3 and R6
Cl or SCN complexes (Figure 1) give HisB10 sites with the
anion positioned on the hexamer three-fold symmetry axis and
coordinated to ZnII.[9–12, 22–24] The structure most relevant to this
work, 1ZNJ,[22] has one HisB10 cavity of HI–R6 that is occupied
both by a Cl coordinated to ZnII and by a phenol. The phenol
ring makes contact with Cl and with the AsnB3, and LeuB6
sidechains in each 3-helix bundle. One PheB1 ring is within 5 
of the phenol ring, one points away, and the third is disor-
dered. The resorcinol-stabilized Cl complex (1EVR) is similar.[23]
A structure with a phenolate ion coordinated to a HisB10 site
was reported,[12, 24] but no coordinates were deposited. The ab-
sence of X-ray structures that show details of an aromatic car-
boxylate bound to the HisB10 cavity of an HI complex likely is
due to disorder arising from the loss of the three-fold molecu-
lar symmetry from placement of a planar ligand into the site.
The planar ligand would be expected to randomly take up any
[a] Dr. M. Bonaccio, D. Niks, Prof. Dr. M. F. Dunn
Department of Biochemistry, University of California at Riverside
Riverside, California 92521 (USA)
Fax: (+ 1) 951-827-4434
E-mail : michael.dunn@ucr.edu
[b] Dr. D. Keidel
The Scripps Research Institute, Department of Molecular Biology
La Jolla, California 92037 (USA)
[c] Dr. N. Ghaderi
Division of Chemistry and Chemical Engineering
California Institute of Technology
Pasadena, California 91125 (USA)
[d] Dr. D. Borchardt
Department of Chemistry, University of California at Riverside
Riverside, California 92521 (USA)
Supporting information for this article is available on the WWW under
http ://dx.doi.org/10.1002/cbic.200800746.
Figure 1. Stereoview of the HisB10 R3 site with SCN
 coordinated to ZnII (green). The hexamer three-fold symme-
try axis (not shown) passes through ZnII and the S, C, and N atoms of SCN . The surface depicts the HisB10 anion
binding site. SCN (van der Waals spheres) AsnB3, LeuB6, CysB7, and HisB10 (sticks) are shown with CPK colors.
The image was created by using PyMOL 1.0 and PDB structure 2TCI.[10]
450  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2009, 10, 450 – 453
one of three symmetry-related positions[4, 8, 21] throughout the
crystal lattice.
19F NMR spectroscopy of fluorinated ligands complexed to
protein sites can be useful for investigating protein structure–
function relationships.[8, 25–27] Four ligands were investigated
herein, 3-trifluoromethylbenzoate (1), 4-trifluoromethylben-
zoate (2), 3-trifluoromethylcinnamate (3), and 4-trifluoro-meth-
ylcinnamate (4). 1D 1H NMR spectra and 1H ACHTUNGTRENNUNG{19F} NOE difference
spectra of R6 HI and the R6 HI variant with B-chain Phe1Glu
and Thr27Glu were measured at pH 8.0 in D2O
[8, 26, 27] at
500 MHz (viz. , Figures 2 and 3). The 1D 1H NMR spectra (Fig-
ure 2 A) and the 1H ACHTUNGTRENNUNG{19F} NOE difference experiment (Figures 2 B
and 3) characterize the complexes of compound 1 formed
with the [D6]phenol-stabilized R6 hexamers of HI (blue traces)
and the HI variant (red traces). The assigned 1H ACHTUNGTRENNUNG{19F} NOE differ-
ence spectra for the aromatic and aliphatic regions are shown
in Figures 2 B and 3, respectively. Chemical shifts of protein res-
onances for HI–R6 complexes with ligand 1 gave excellent
agreement (0.05 ppm) for most residues with the values that
were reported for the R6 complex with the HisB10 ligand, 3-
nitro-4-hydroxybenzoate (5).[21] Except for differences due to
the amino acid substitutions, the resonances of the HI variant
complex gave similar agreement.
The 1D 1H NMR spectra (Figure 2 A) include peaks in
the 7.3 ppm to 8.3 ppm region for free ligand (sharp) and
bound ligand (broad, slightly shifted) ; this is consistent
with moderately slow to slow exchange.[8, 21] The signature
protein peaks at 5.0 ppm to 6.6 ppm (Figure 2 A) establish that
both the HI and the variant complexes have the R6 conforma-
tion.[13–15, 18–21, 28–30] The complexes with compounds 2–4 gave
similar results (data not shown).
The 1H ACHTUNGTRENNUNG{19F} NOE difference spectra (Figures 2 B and 3) al-
lowed us to identify protein residues within ~5  of the CF3
fluorine atoms of bound 1. By comparison of chemical shift
values with the assigned resonances of the HI–R6 complex
with 5,[21] assignments of the strong peaks were made for the
complex of HI–R6 with 1. The ring protons of PheB1 give reso-
nances at 7.31 and 7.34 ppm in the complex with 5, and the
corresponding complex with Cl gives PheB1 resonances at
7.30 and 7.34 ppm.[21] The NOEs in Figure 2 B at 7.13 and
7.20 ppm in HI are completely missing in the HI variant R6 dif-
ference spectrum, whereas there is a good correspondence be-
tween NOEs observed in HI and in the HI variant elsewhere in
the spectrum. Because the variant has PheB1 replaced by Glu,
the absence of NOEs between 7.0 and 7.28 ppm in the variant
must be due to the substitution of Glu for Phe; this confirms
that the 7.13 and 7.20 ppm signals in the HI complex arise
from NOEs between the fluorine atoms of the CF3 group of 1
and the PheB1 Hd and He atoms (slightly shifted by ligand
ring current effects). The HI–R6 complexes with compounds 2–
4 also show strong NOE interactions at 7.28 ppm that are simi-
lar to the 7.20 ppm peak in the complex with 1.
In the aliphatic region, the HI variant and HI–R6 complexes
with 1 give similar NOEs with chemical shifts assigned to the
protons of LeuB6 and AsnB3 (Figure 3) based on the assign-
ments for the HI complex with 5.[21] Because the binding locus
is not in question,[8, 15, 16, 19–21] a more rigorous assignment
method was not needed. Control experiments demonstrated
that spin diffusion was not responsible for any of the observed
NOEs (see Figures S1–S3 in the Supporting Information).
The NOEs observed between the CF3 fluorine atoms of com-
pounds 1–4 and the PheB1 rings were unexpected. In most X-
ray structures of HI–R6, the PheB1 rings extend out into solu-
tion >6  away from the opening to the HisB10 site. Because
there are no published structures of aromatic carboxylates
bound to the HisB10 site, we decided to explore the binding
of ligands 1–4 by modeling their structures into the HisB10
site of the HI–R6 hexamer, 1ZNJ.
[22] Figure 4 shows a structural
model based on the coordinates for 1ZNJ that is consistent
with the observed NOEs for the HI complex with 1. This model
was generated as follows: the coordinates for Cl and phenol
at the HisB10 site in 1ZNJ were replaced by 1, hydrogen atoms
were added to the protein, and crystallographic water, andACHTUNGTRENNUNGdisordered protein residues were replaced prior to simulation.
Minimization was performed by using the Discover (CVFF force
field) and Discover_3 (ESFF force field) modules present in the
molecular modeling 3D graphical interface, Insight II.[31–34]
Both water and protein objects were minimized in turn by
using a Cell-Multipole summation, then the “steepest descents”
minimization algorithm, and finally the Polak–Ribiere Conju-
Figure 2. A) 1H NMR spectra comparing R6 complexes of 1 with human in-ACHTUNGTRENNUNGsulin (blue) and with the human insulin variant with B-chain Phe1Glu and
Thr27Glu (red). B) Aromatic region of the 1HACHTUNGTRENNUNG{19F} NOE difference spectra for
R6 human wild-type (blue) and R6 variant (red) complexes with 1.
Figure 3. Aliphatic region of the 1HACHTUNGTRENNUNG{19F} NOE difference spectra for R6 human
insulin (blue) and the human insulin variant (red) complexes with 1. Spectra:
average of 16 000 scans, mixing time = 0.9 s, acquisition time = 2.047 s, relax-
ation delay = 1.5 s.
ChemBioChem 2009, 10, 450 – 453  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 451
gate Gradient minimization (convergence value = 0.10).[31–34]
The model (Figure 4) shows 1 coordinated to the HisB10 ZnII in
one of three symmetry-related positions. In this model, the H
atoms of LeuB6, AsnB3, and the PheB1 ring are within 5  of
the fluorine atoms of CF3. Ligand exchange among the three
symmetry-related positions would allow NOEs to each of the
three LeuB6, AsnB3, and PheB1 residues that form the walls of
the cavity.
These NOE experiments establish that the complex of 1 with
HI–R6 takes up a conformation in which the PheB1 ring pro-
tons are located within 5  of the fluorine atoms present on
the CF3 group. The structural model (Figure 4) predicts that 1
makes van der Waals contact with the ring of a PheB1 in a
structure where this PheB1 and the AsnB3 residues cap the
HisB10 anion-binding site. Structural models also were calculat-
ed for compounds 2–4. These models show a similar capping
of the HisB10 site (Figure S4). The model structures suggest
possible strategies for the design of aromatic carboxylates as
allosteric effectors to stabilize R-state HI hexamers for use in
formulations to treat diabetes.
Experimental Section
ZnII-free human insulin and the insulin variant with B-chain
Phe1Glu and Thr27Glu were gifts from Novo Nordisk. All other
chemicals were purchased from Sigma–Aldrich and used without
further purification. Samples of zinc–insulin hexamers were pre-
pared for NMR spectroscopy as was previously described,[8] byACHTUNGTRENNUNGincubation of the proteins in D2O so that the only resonancesACHTUNGTRENNUNGobserved were from 1 and the non-exchangeable/very slowly ex-
changing protons of the proteins. The pulse sequence used for
these experiments gave the 1HACHTUNGTRENNUNG{19F} NOEs directly. Alternate scans
were subtracted to leave the NOE. Consequently, only half the
scans lead to accumulation of NOE signals with the other half pro-
viding a correction for T1 relaxation during the mixing time. This
sequence can easily detect NOEs in the 0.1 % range. The observa-
tion of NOEs was critical to this study, not their quantification;
however, NOEs observed here are estimated to be in the 1–2 %
range.
Acknowledgements
We thank Novo Nordisk for the
wild-type human and variant in-
sulin, and for a gift to M.F.D. in
support of this work.
Keywords: 19F NMR ·
allosterism · insulin · molecular
modeling · NMR spectroscopy ·
nuclear Overhauser effect
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452 www.chembiochem.org  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2009, 10, 450 – 453
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Received: November 12, 2008
Published online on January 14, 2009
ChemBioChem 2009, 10, 450 – 453  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 453


^1H{^(19)F} NOE NMR Structural Signatures of the Insulin R_6 Hexamer: Evidence of a Capped HisB10 Site in Aryl- and Arylacryloyl-carboxylate Complexes

https://authors.library.caltech.edu/15713/1/Keidel2009p64910.1002cbic.200800746.pdf

^1H{^(19)F} NOE NMR Structural Signatures of the Insulin R_6 Hexamer: Evidence of a Capped HisB10 Site in Aryl- and Arylacryloyl-carboxylate Complexes

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