Spectroscopic evidence for preexisting T- and R-state insulin hexamer conformations

PROTEINS Structure, Function, and Genetics 26377-390 (1996)

Spectroscopic Evidence for Preexisting TI and R-State Insulin Hexamer Conformations Wonjae E. Choi,' Dan Borchardt? Niels C. Kaarsholm: Peter S. BrzoVic,l and Michael F. Dunn'* 'Departments of Biochemistry and 'Chemistry, University of California, Riverside, Riverside, California; 3 N ~ ~ ~ Research Institute, Novo Nordisk AIS, Bagsvaerd, Denmark

ABSTRACT The insulin hexamer is an allosteric protein exhibiting both positive and negative cooperative homotropic interactions and positive cooperative heterotropic interactions (C. R. Bloom et al., J. Mol. Biol. 245, 324 330,1995). In this study, detailed spectroscopic analyses of the UVNis absorbance spectra of the Co(I1)-substituted human insulin hexamer and the 'H NMR spectra of the Zn(I1)-substituted hexamer have been carried out under a variety of ligation conditions to test the appli- cability of the sequential (KNF) and the half-site reactivity (SMB) models for allostery. Through spectral decomposition of the characteristic d

d transitions of the octahedral Co(I1)-T-state and tetrahedral Co(I1)-R-state species, and analysis of the 'H NMR spectra of T- and R-state species, these studies establish the presence of preexisting T- and R-state protein conformations in the absence of ligands for the phenolic pockets. The demonstration of preexisting R-state species with unoccupied sites is incompatible with the principles upon which the KNF model is based. However, the SMB model requires preexisting T- and R-states. This feature, and the symmetry constraints of the SMB model make it appropriate for describing the allosteric properties of the insulin hexamer. Proteins 26377490 o 1996 Wiley-Liss, Inc.

Key words: allostery; hexameric insulin; positive and negative cooperativity; half-site reactivity

INTRODUCTION Allosteric proteins regulate and coordinate phys-

ical and chemical processes in living systems through cooperative ligand binding interactions that modify enzyme activity and/or protein function. Recent studies have shown that the insulin hexamer exhibits positive heterotropic and positive and negative homotropic allosteric ligand binding pr~per t ies . l*~-~ The phenol-induced T- to R state allosteric transition of the insulin hexamer has been well characterized in the crystalline and in s o l ~ t i o n . ~ , ~ - ~ , ~ ~ " ~ Crystal structures of the T,, T3R3, and R, Zn(I1) hexamers show that the conformational transition involves conversion of residues Bl-B8 of each mono-

0 1996 WILEY-LISS, INC.

mer from an extended chain (T-state) to a-helix (R- state)10,13J4 (Fig. 1). Upon conversion to R,, six essentially identical amphipathic binding pockets are created at the subunit interfaces. The binding of phenolic molecules to these pockets involves hydro- gen bonds between the ligand hydroxyl and the car- bony1 oxygen of CysA6 and the amide nitrogen of CysAll and favorable van der Waals' interactions between the ligand ring and several side chains within the pocket. In the T, conformation, these pockets are filled by the LeuB6 side The conformational transition forces the coordination geometry of the Zn2+ ions at the two HisBlO sites to change from octahedral (T,) to pseudotetrahedral (R,) (Fig. 1). The octahedral Zn2+ centers are coordinated by the three HisBlO imidazole ring nitrogens and by three water molecules.s The pseudotetrahedral Zn2+ centers are coordinated by the same three HisBlO nitrogens and a fourth ligand from s o l ~ t i o n . ~ ~ , ~ ~ , ~ ~ This fourth (exchangeable) ligand position can be occupied by a wide range of

form of the hexamer consists of interdigitated T, and R, units. The R, units are comprised of a tetrahedral HisBlO metal site and three phenolic pockets; the HisBlO site of the T, unit usually is octahedral, but in some instances may be tetrahedral in the crystalline s t a t e . l l ~ ~ ~

The two most widely applied models to describe negative cooperative allosteric behavior are the se-

anionic and neutral ligands.4*6,7,15,17,22 Th e T,R,

Abbreviations: T and R are used throughout to designate insulin forms with extended (T) and OL helical (R) conformations of B-chain residues 1-8. T6, T3R3, and R, designate the three general conformation states of the insulin hexamer. T,T,' and R,R,' designate conformations with one threefold axis and three pseudo-twofold axes of symmetry, while T," R3' designates the hexamer with only a single, threefold, axis of symmetry' . Co(II)- and Zn(II)-T,, -T3R3 or -Re, designate Co(I1) or Zn(I1)-substituted insulin hexamers. KNF, the sequential model for allostery'; SMB, the half-site reactivity (suboptimal symmetry) model for c~operativity~

Dr. Choi's current address is Joslin Diabetes Center, One Joslin Place, Boston, MA 02215; Dr. Bmvic's current address is Department of Biochemistry, University of Washington, Se- attle, WA 98195.

*Correspondence to: Michael F. Dunn, Department of Bio- chemistry, University of California, Riverside, CA 92521.

Received 6 June 1996; accepted 7 June 1996.

378 W.E. CHOI ET AL.

N

Fig. 1. Ribbon diagramsz3 showing the structural differences between the subunits of the T, (A,a) and the phenol bound R, (B,a) hexamers together with the octahedral coordination of the T-state HisBlO site (A,b) and the tetrahedral coordination of the R-state HisBlO site (B,b). In (B), the molecule of phenol indicates the location of the phenolic pocket between the two a-chain heli-

ces. In (C), trimeric units of T, (a) and R, (b) viewed along the threefold axis are shown as Ca line drawings of the backbone. The side chains of HisBlO and GIuB13 are shown in (a) and (b), and in (b) the positions of the three phenol molecules (P) asso- ciated with this R, unit are also indicated. From Bloom et aI.,’with permission.

PREEXISTING ALLOSTERIC STATES IN INSULIN 379

quential model2 (the KNF model, Figure 2A) and the half-site reactivity (the SMB model, Figure 2B). The KNF model proposes that ligand- induced site-site interactions between subunits in- duce sequential conformational changes within the protein. The KNF model is flexible with at least m conformations (where m = the total number of ligand binding sites), and a unique ligand dissocia- tion constant for each conformation. Although this model can explain both positive and negative homotropic interactions, the number of parameters in the system makes data analysis and verification of the model difficult. Furthermore, in the KNF model, since ligand-induced positive or negative conformational effects within adjacent sites are transmitted through different binding domains, there is no clear- cut distinction between positive and negative coop- e r a t i ~ i t y . ~

The SMB model postulates that both positive and negative cooperativity and half-site reactivity can arise from a painvise heterogeneity in sites due to conformational isomerization between quaternary states with suboptimal ~ y m m e t r y . ~ , ~ ~ A suitably tailored SMB model has been shown to be capable of describing the positive heterotropic and positive and negative homotropic ligand binding properties of the Co(I1)-substituted insulin hexamer (Fig. 2B).5-7

To further examine the mechanism of the insulin allosteric transition, we present herein a detailed spectroscopic analysis of metal-ligand interactions in the Co(I1)-substituted insulin hexamer. The results of these studies provide evidence consistent with the existence of preformed R-state binding sites for phenolic ligands, a finding consistent with predictions of the SMB model but which contradicts the KNF model.

MATERIALS AND METHODS Materials

The chemicals employed in these studies were re- agent grade or better and were used without further purification. Metal-free human insulin was supplied by Novo Nordisk (Denmark). Phenol was purchased from J.T. Baker Inc. Trizma base and sodium azide were purchased from Sigma. Potassium thiocyanate, perchloric acid, zinc sulfate, and calcium sulfate were purchased from Mallinckrodt. Sodium chloride was purchased from Fisher Scientific. Cobalt per- chlorate was purchased from Alfa Products. 'H2S04, 40% Na02H, d,-phenol, and 'H20 were purchased from Aldrich.

Methods Insulin monomer concentrations were determined

from absorbance measurements at 280 nm, using a value2, of E,,, = 5700 M-lcm-l. Co(I1)- substituted insulin hexamers used in UV-Visible spectroscopic studies were prepared as previously described?

Electronic spectra (absorption and derivative spectra) were recorded on a Hewlett-Packard 8452A Di- ode Array Spectrophotometer. Sample preparation and spectroscopic parameters for the 'H NMR studies were as previously described., One-dimensional PRESAT spectra were collected on a GN-500 spec- trometer with a Nicolet 1280 computer. Spectral analysis of NMR data were carried out on the software FELIX installed in a Silicon Graphics computer system. The nonlinear curve-fitting software program, Peakfit (Jandel Inc.), was used to fit both the absorption (gaussian functions) and the 'H NMR spectra (lorentzian functions).

RESULTS Monovalent Anion-Stabilized Hexamers Show a Mixture of T- and R-State Spectral Characteristics

Co(I1) substitution has been widely used as an op- tical technique to study the structural transitions of a variety of Zn(I1)-containing protein^.'^,^^ For tetrahedral complexes, the visible spectrum derived from the d +- d transitions is dominated by relatively intense bands in the 550 to 750 nm range. The visible spectrum of the octahedral complex is dominated by an envelope of lower intensity centered at 500 nm.29-31 The interconversion of octahedral and tetrahedral coordination geometries during the T- to R-state transition (Fig. 1) significantly alters the visible spectrum of the Co(I1)-substituted insulin hexarner.4,5,15.17.21,32 The octahedral Co(I1)-T, complex (Fig. 3, spectrum a) exhibits a broad d += d envelope of low intensity (Amm = 495 nm, E,,, = 45 M-lcm-'), whereas the tetrahedral Co(I1)-R, complexes (spectra b and c) gives much more intense d + d bands (h,,, 540-630 nm, E,, > 300 M-lcm-l) that are very sensitive to the identity of the fourth (exchangeable) ligand. For strongly coupled d + d transitions in inorganic Co(I1) complexes, the absorption bands are well described by symmetric gaussian-shaped functions.33 The overlapping spectral bands in each of the Co(I1)-hexamer complexes (Fig. 3) arise from different Co(I1) electronic transitions, but the correlation between the number of gaussian functions needed to fit the spectra and the total number of electronic transitions is not clear. To assist determination of the number of gaussian functions needed to suitably fit the visible region of the Co(I1)-spectra, derivative spectra (Figs. 4 and 5) were collected and analyzed. Although derivative spectroscopy does not necessarily identify the exact number and the positions of all the component bands within the absorption profile, the second and fourth derivations can identify the apparent positions of composite bands34 (Fig. 4AB). It was found that reasonable empirical fits of the d -+ d transition profiles of the various Co(I1)-substituted insulin hexamer complexes were possible using a relatively small


A L 1

11

T=O It p 11 p

Fig. 2. (A) The KNF sequential model for allostery2 for a hexamer of identical subunits. The conformational transition from the T-state (circles) to the R-state (squares) is induced by ligand binding, consequently, a preexisting equilibrium between unliganded T- and R-states is not a feature of the model. (B) A modified SMB model for all~ste$.~-' tailored to the insulin hexamer is shown. From Brzovic et al.,'with permission. The allosteric constants, LoA and LoB, describe the preexisting equilibria between allosteric forms. LA and LoB are determined by the amount and the type :f anions present in solution. Due to the unfavorable value of Lo , without phenolic ligands present, the overall allosteric transition is restricted to the equilibrium between the T3T3' and the T," R,' states. When phenolic ligands are present, two possible events

number of symmetric gaussian functions. The gaussian function used in this study is

flu) = f , exp (-In Z[Z(u - u,)/f12)

where f , is the band height a t its maximum band position (u,), and H is the bandwidth at half-height. Decomposition of the absorption profiles by these

could occur, (1) a new equilibrium is established between the T,"R," and the R,R,' states and (2) phenolic ligands bind in the empty protein pockets of the T,"R," state. Whether the new equilibrium between the T,'R,O and the R,R,' states is established before the formation of the T,"R,"P, complex, is under investigation. The phenol binding to the empty protein pockets of the T3W3' state must precede the structural transition from the T30R30 state to the R,R,' state because the binding process does not require a conformational transition. Once the R,R,'P,complex is formed, the formation of the final P,R,R,'P, complex involves the rapid binding of the phenolic ligand to the three empty pockets of the new R, trimer.

gaussian functions allows the examination of three properties of each composite gaussian band: the wavelength position (u,), the width at half-height (H), and the intensity Cf,).

Figure 4A shows the visible spectrum of the Co(I1) d + d transition of the C1--coordinated Co(I1)-R- state hexamer obtained in the presence of phenol (a),


400 600 800 Wavelength (nm)

Fig. 3. Electronic absorption spectra of Co(ll)-substituted insulin hexamers: the H,O-coordinated octahedral Co(ll) complex (the T, state) (a); the phenolate-coordinated tetrahedral Co(ll) complex (an Re state measured in the presence of 60 mM phenol) (b); and the CI--coordinated tetrahedral Co(ll) complex (an R, state measured in the presence of 60 mM phenol and 100 mM NaCI) (c).

the first (b), the second (c), and the fourth (d) derivations of the spectrum. The visible spectrum is characterized by an intense envelope with A,, at 580 nm and a shoulder at 550 nm. Casual inspection indicates the d + d transition envelope consists of two composite bands. The first derivation shows an inflection point with an x intercept a t 580 nm and two maxima and one minimum at the following wavelengths; 540, 570, and 620 nm, respectively. The second derivation is more informative; the maxima and the minima of the first derivation become the x intercepts, while three large new minima de- velop at 548,574, and 606 nm. In the fourth derivation, the previous minima are now maxima with two smaller maxima at 506 and 640 nm. These results imply that the d + d transitions of the Cl-coordinated Co(I1)-R-state can be fitted by three to five gaussian functions.

Figure 4B shows the envelope of d + d transitions for the SCN-coordinated Co(I1)-R-state hexamer (a), along with the first (b), the second (c), and the fourth (d) derivation of the spectrum. The first derivative spectrum shows an x intercept at 572 nm, a minimum at 606 nm and two maxima at 530 and 560 nm. The second derivative spectrum shows four minima at the following wavelengths: 536,570,594, and 606 nm. The fourth derivation shows seven maxima at the following wavelengths: 536, 570, 594, 606, 630, 646, and 660 nm. The maximum at 660 nm is due to noise from the initial spectrum (see spectrum a). These results indicate that the SCN-coordinated Co(I1)-R-state spectrum can be fitted by four to six gaussian functions. Table I shows the results obtained for other ligand-coordinated Co(I1)-insulin hexamers.

Figure 5Aa shows the gaussian spectral decomposition obtained for a 0.34 mM solution of the Co(I1)- substituted insulin hexamer in the presence of 20

mM phenol and 500 mM NaCl. The spectrum of the C1- complex is characterized by an intense envelope with A,, = 580 nm. This spectrum is well fitted by six gaussian curves with the following peak centers: 502 (11,534 (2), 548 (31, 580 (4), 606 (9, and 624 nm (6) (Fig. 5Aa). Relative area calculations show that most of the area under the envelope is accounted for by curves 3, 4, 5, and 6. Based on the peak centers and the intensities, these six gaussian functions correspond to a mixture of electronic transitions consisting of octahedral and tetrahedral Co(I1) com- p l e ~ e s . ” - ~ ~ We have previously reported that the SCN-mediated Co(I1)-insulin hexamer shows a spectral profile consisting of a mixture of two species, an octahedral and a tetrahedral Co(I1) ~omplex .~

Figure 5Ab shows the gaussian decomposition of the d --* d transition envelope for a 1.02 mM Co(I1)- substituted insulin hexamer in the presence of 1.0 M NaCl. This envelope is made up of a low-intensity, broad-band centered at 500 nm with an even lower intensity shoulder a t 590 nm. Based on the intensity and the envelope maximum, this spectrum is dominated by a Co(I1)-T-state hexamer with octahedral c~ordination.~ Although the overall profile is that of the T-state, gaussian curve fitting of this d + d envelope shows that the spectrum can be fitted by the same six gaussian curves used to fit the C1--coordinated R-state adduct spectrum. In Figure 5Ab, the 500 nm gaussian curve is assigned to an octahedral Co(I1) complex. The small deviation from this gaussian on the long wavelength side suggests the presence of additional transitions. Figure 5Ac shows the gaussian decomposition of the d + d envelope for a 1.02 mM solution of the Co(I1)-substituted insulin hexamer in the absence of salt. For comparative pur- poses, the same six gaussian functions were used to describe Figure 5Aa, Ab, Ac. As in Figure 5Ab, the major component of the spectrum in Figure 5Ac is characteristic of the octahedral Co(I1) T-state, but unlike the spectrum in Figure 5Ab, the shoulder a t 590 nm has disappeared. The dominant gaussian curve shown in Figure 5Ac is located at 500 nm, and comprises about 95% of the total relative area under the spectrum (Table I). In the absence of NaCl (Fig. 5Ac), the relative areas under curves 2-4 are about 4% of the total area, while the respective relative area in the presence of 1M NaCl is about 8% (Table I).

With and without C1- ions, the major component of the overall spectrum arises from the octahedral (T-state) Co(I1) complex (curve 1 in Table I). Adding C1- alters the intensities and the relative areas of curves 3 to 6, but does not dramatically alter those of curves 1 and 2. Unlike the T-state spectrum, the major components of the Co(I1)-R-state spectrum arise from the pseudotetrahedral Co(I1) complex (gaussian functions 3 to 6). In the phenol complex, the relative intensities and the peak areas of curves 3 to 6 are dramatically increased. These data estab-

W.E. CHOI ET AL.

I I

A

I V I

400 500 600 700 Wavelength (nm)

Fig. 4. A: The visible spectrum of the Co(ll) d -+ d transitions of the CI--coordinated Co(ll)-R-state hexamer (a), the 1st (b), the 2nd (c), and the 4th derivation of the spectrum (d). The visible spectrum is characterized by an intense envelope with A,, at 580

lish that curves 1 and 2 are characteristic of the octahedral Co(I1) complex, while curves 3 to 6 are characteristic of the tetrahedral Co(I1) complex.

The R, Units of T,R,-SCN- and the Phenol Induced Re-SCN- Complexes Have Identical Co(I1) Coordination Geometries

Figure 5B shows the gaussian decomposition for the spectrum of the Co(I1)-insulin hexamer formed in the presence of 640 mM SCN- (a), and in the presence of 400 mM SCN- and 100 mM phenol (b). Previous works from this laboratory have shown that SCN- stabilizes the T,R3 hexamer in s~lut ion.’*~-~ Figure 5B shows that the Co(I1) d + d transitions of the SCN--Co(II)-insulin hexamer are well fitted by seven gaussian functions. Table I shows that gaussian functions 1 and 2, which make up about 22% of the total relative area under the spectral envelope in Figure 5Ba, correspond to an octahedral Co(I1) com-

400 500 600 700 Wavelength (nm)

nrn and a shoulder at 550 nrn. B: The d -+ d transition envelope of the SCN--coordinated Co(ll)-T3R3 hexamer (a), the 1 st (b), the 2nd (c), and the 4th derivation of the spectrum (d).

plex. Figure 5Bb shows that the spectral profile of the phenol-induced, SCN-coordinated Co(I1) R-state hexamer is composed of the essentially identical seven gaussian functions. The differences reside only in the peak intensities (and the relative areas) of the gaussian functions. In Figure 5Bb, the gaussian functions for the octahedral Co(I1) component (curves 1 and 21, only make up 7.3% of the total relative area under the Co(I1) d + d transition envelope.

‘H NMR Spectra of Insulin Hexamers Provide Evidence for Preexisting R-State Conformations

‘H NMR spectra provide additional evidence that anions stabilize an “R-state like” conformation. Brzovic et a1.6 have shown that in solutions with high concentrations of simple inorganic anions (e.g., C1-, SCN-, N3-, and I-), the ‘H-NMR spectra con-


Fig. 5. A: (a) Gaussian decomposition of the d -+ d transition envelope of a 0.34 mM Co(ll)-substituted insulin hexamer in the presence of 20 mM phenol and 500 mM NaCI. The d - d bands of the CI--coordinated Co(ll)-R-state hexamer are characterized by an intense envelope with a A,, at 580 nm. (b) Gaussian decomposition of the d -+ d transition envelope of a 1.02 mM Co(ll)-substituted insulin hexamer in the presence of 1M NaCI. Note the arrow indicating the increase in relative area compared

0.40

0.20

0

0.30

500 600 Wavelength (nm)

tain signatures that are similar to those characteristic of the Zn(I1)-T3R3 conformation. Figure 6A shows the aromatic regions of the 'H NMR spectra of the Zn(I1) insulin hexamer in the presence of 3-nitro-4-hydroxybenzoate (a), SCN- (b), N3- (c) I- (d), C1- (e), and in the absence of any added ligands (f). Previous works from this lab~ratory,~,~, ' , '~, have shown that T3R3 and R6 hexamers have distinc- tively characteristic 'H resonances in the aromatic region between 5.0 ppm and 8.0 ppm. These resonances have been given sequence-specific assign- men@; for example, the resonance at 6.5 ppm arises from the 3,5 aromatic protons of TyrB16, while the resonance at 6.6 ppm arises from the 3,5 aromatic protons of TyrB26. In the T-state spectrum (f), the aromatic region (6.5-8.0 ppm) consists of a broad envelope arising from overlapping resonances from the aromatic protons of four Tyr, three Phe, and two

500 600 700 Wavelength (nm)

to spectrum (c). (c) Gaussian decomposition of the d -+ d transition envelope of a 1.02 mM Co(ll)-substituted insulin hexamer in the absence of any added ligands. B: Gaussian decomposition of the d + d transition envelopes of the Co(ll)-T,R,-insulin hexamer in the presence of 640 mM SCN- (a) and the Co(ll)-Re-insulin hexamer in the presence of 400 mM SCN- and 100 mM phenol (b).

His residues/insulin subunit. There are also two smaller resonances at 7.7 and 7.8 ppm, which have been assigned to the ring C-2 protons of the HisB5 and HisBlO r e s i d ~ e s . ~ , ~ , ' ~ , ~ ~ In the SCN--induced spectrum (b), many resonances are significantly sharpened, and the resonances assigned to the ring protons of TyrB16 and TyrB26 are shifted upfield to 6.5 and 6.6 ppm, respectively. The upfield shifts of these resonances signal the formation of a T3R3 species in s o l ~ t i o n . ~ , ~ , ' ~ The resonances due to the c-2 protons of HisB5 and HisBlO also have shifted and sharpened. Similar, but slightly broadened Tyr resonances are present in the N3-- and (c), I--induced spectra (d). In the 3-nitro-4-hydroxybenoate-induced (a) and Cl--induced spectra (el, the presence of R-state resonances is not immediately obvious, but spectral decomposition by lorentzian functions (Fig. 6B) give evidence of the two Tyr resonances at


TABLE I. Gaussian Decomposition of the d + d Electronic Transitions of the Co(I1)-Insulin Hexamer

Pocket Metal Peak Peak Relative Band Area % ligand ligand no. center (nm) intensity width(nm) Area Phenol"

Noneb

Nonec

Noned

Phenol'

c1-

c1-

None

SCN-

SCN-

1 502 2 534 3 548 4 580 5 606 6 624 1 500 2 530 3 548 4 586 5 608 6 634 1 500 2 530 3 546 4 584 5 592 6 622 1 504 2 526 3 538 4 568 5 596 6 610 7 616 1 504 2 524 3 540 4 568 5 596 6 610

0.025 0.033 0.086 0.215 0.091 0.034 0.060 0.002 0.002 0.006 0.003

<0.001 0.064 0.003 0.002 0.003

>0.001 >0.001

0.061 0.043 0.113 0.279 0.225 0.018 0.058 0.020 0.045 0.172 0.363 0.291 0.031

29.11 13.05 10.55 16.99 10.92 20.08 40.02 7.99

12.72 18.44 16.92 7.44

37.6 8.3

10.4 19.0 6.9

14.5 39.9 12.4 10.2 14.7 13.6

21.8 29.2 11.8 10.8 14.0 13.6 6.9

6.20

1.40 1.07 2.26 9.16 2.50 1.69 5.58 0.05 0.08 0.28 0.12 <0.01 5.46 0.07 0.04 0.16

>0.01 0.03 5.60 1.35 2.90

10.30 7.67 0.28 3.51 1.17 1.34 4.64

9.90 0.05

12.8

7.7 5.9

12.5 50.7 13.8 9.3

91.3 0.8 1.3 4.5 2.0 0.1

94.7 1.2 0.7 2.8

>0.1 0.6

17.9 4.3 9.3

32.9 24.5 0.90

11.10 3.4 3.9

13.5 37.1 28.8

1.5 7 616 0.076 21.5 4.10 11.8

"Fit for Figure 5Aa. bFit for Figure 5Ab. %it for Figure 5Ac. dFit for figure 5Ba. 'Fit for Figure 5Bb.

6.6 and 6.5 ppm, indicative of a trace of the T3R, species.

The lorentzian decomposition of the partial aromatic regions (6.75 - 6.25 ppm) of 'H NMR spectra of the Zn(I1) insulin hexamers in the presence of 3-nitro-4-hydroxybenzoate (a), SCN- (b), N,- (c), I- (d), and C1- (e) are shown in Figure 6B. Spectrum (f) is that of the Zn(I1)-insulin hexamer with no added ligands. The partial aromatic region shown in spectrum b is well fitted by four lorentzian functions with the following peak centers; 6.73,6.68,6.61, and 6.49 ppm. The relative areas of the four lorentzian functions represent the relative amounts of protons under each respective resonance. The slightly broadened resonances in spectrum c, collected in the presence of N3-, also can be fitted by the same four lorentzian functions used to describe spectrum b. Of the anions studied, the pseudohalides, SCN- and

N3-, are more effective in stabilizing R-state species than is 3-nitro-4-hydroxybenzoate or the halides, I- and C1-. Calculations of the relative areas under the TyrB16 resonance (6.5 ppm) show that N3- yields about 62% R-state while I- and C1- yield 38% and 13%, respectively. Due to the presence of resonances from the unbound form of 3-nitro-4-hydroxybenzoate, a relative area calculation for this ligand was not made.

'H-NMR Spectra of the Phenol-Induced T, to R, Conformational Transition

The aromatic resonances provide useful 'H-NMR signals with which to monitor the T, to R, phenol- induced allosteric transition of the insulin hexamer. Of particular utility are the resolved signals which arise from the HisB5 C-4 proton and the 3,5 protons of TyrBl6 and TyrB26 in the R-state conforma-

385 PREEXISTING ALLOSTERIC STATES IN INSULIN

7.0 6.0 PPM

Fig. 6. A: The aromatic regions of the ’H NMR spectra of the 0.5 mM Zn(ll)-insulin hexamer in the presence of 2 mM 3-nitro-4- hydroxybenzoate (a), 50 mM SCN- (b), 50 mM N,- (c), 50 mM I- (d), 50 mM CI- (e), or in the absence of any added ligands (9. B: The lorentzian decomposition of the partial aromatic regions (6.7 to 6.25 pprn) of ‘H NMR spectra of 0.5 mM Zn(ll) insulin hexamer in the presence of 2 mM 3-nitro-4-hydroxybenzoate (a), 50 mM

tion4,6,15 (Fig. 7). The upfield position of the TyrB16 resonance is due to the anisotropic ring current effects of HisB5 and TyrB26, which form a set of stack- ing interactions only in the R-state6J0J3J4 (Fig. 8). The upfield shifted position of the HisB5 C-4 ring proton is due to a similar set of aromatic ring interactions with TyrB16 and the bound phenolic ligand

6.75 6.50 6.25 PPM

SCN- (b), 50 mM N,- (c), 50 mM I- (d), and 50 mM CI- (e). Spectrum (f) is that of the Zn(ll) insulin hexamer with no added ligands. The region of the spectra shown was well fitted by four lorentzian functions with the following original peak centers; 6.73, 6.68, 6.61, and 6.49 ppm. Note the gradual downfield shift of the TyrBl6 and the TyrB26 resonances located at 6.49 and 6.61 pprn, respectively.

(Fig. 8). Therefore, these aromatic proton resonances provide signals characteristic of both the T- to R-conformational transition of the insulin hexamer, and the binding of ligand to the phenolic pocket.

Previous work from this laboratory has shown that the T, to R, conformational transition of the


I I

7.0 6.0 PPM

Fig. 7. 'H-NMR spectra at 500 MHz in 'H,O showing the aromatic region of the zinc insulin hexamer spectrum as a function of the concentration of d,-phenol measured at a pH meter reading of 8.0 and 25". Conditions: the concentrations of d,-phenol are as follows: (a) 0, (b) 0.5, (c) 1.0, (d) 2.0, (e) 3.0, (9 4.5, (9) 7.0, (h) 20 mM. [Insulin monomer] = 3 mM; the [Zn2+]: [Ca"]: [insulin monomer] ratio = 2: 1.6 : 6, and [NaCl] = 50 mM. Spectra (a) and (h) correspond respectively to the T6 and R, species. Spectra (9 and (9) are dominated by the T,R, species.'

Zn(1I) insulin hexamer is a three state process which involves a T,R, intermediate s p e c i e ~ ~ , ~ , ~ (Fig. 2). During the T, to T,R, component of the transition, most protein resonances appear to exhibit slow exchange behavior on the NMR time scale. The 3,5 proton resonance of TyrB26 in the R-state (6.67 ppm) does not appreciably change linewidth or chemical shift during the course of the titration (Fig. 7). Only the area of the B26 resonance changes markedly upon addition of phenol. In contrast, the C-4 resonance of HisB5, a residue which forms part of the phenolic binding pocket, undergoes extensive line broadening, particularly at low phenol concentrations, and a large upfield chemical shift pertur- bation, indicative of a resonance in intermediate to fast exchange. The 3,5 proton resonance of the TyrB16 side chain undergoes both an upfield chemical shift and an increase in intensity with increas- ing phenol concentration. This resonance is likely

influenced both by ligand binding, and by the slow protein conformational transition. The T,R, to R6 conformational transition is dominated by very slow exchange behavior, as is particularly evident for the TyrB16 and HisB5 resonances. Both signals exhibit defined peaks separated by less than 50 Hz.

DISCUSSION The insulin hexamer is a new example of an al-

losteric p r ~ t e i n . ' ~ ~ , ~ . ' ~ The refined, high resolution x-ray structures of Zn(I1)-T,, Zn(I1)-T,R,, and Zn(I1)-R, render the insulin hexamer one of a small number of allosteric proteins for which there is detailed high resolution structural information on both the T- and the R - s t a t e ~ ~ - ' ~ ~ ~ ~ (Figs. 1 and 8). This structural information provides an essential frame- work for consideration of the relationship between the structure of the hexamer and its allosteric properties.

The Evidence Supports an SMB Model for the Insulin Hexamer

Both the KNF and the SMB model^^,^ (Fig. 2) can explain positive and negative homotropic interactions within the same oligomer. However, only the SMB model places symmetry constraints on allosteric transitions, and only the SMB model predicts preexisting equilibrating mixtures of the T- and R-states. The SMB and KNF models make very different predictions about the expected spectroscopic properties of the insulin hexamer. The "pairwise asymmetry in subunit conformation", of the SMB model assumes that (1) the suboptimal symmetry can lead to site heterogeneity, (2) there can be conformational isomerization among quaternary states of optimal and suboptimal symmetries, and (3) T- and R-state species can preexist, hence empty ligand sites can also p r e e x i ~ t ~ ? ' ~ (viz., Figure 2B). This model predicts that half-site reactivity is a consequence of oligomer assembly from asymmetric dimeric units and that both positive and negative cooperativity can arise from certain combinations of the model parameters. The SMB model elaborated for the insulin hex- amer7 (Fig. 2B) predicts three conformational states of the insulin hexamer, T,T,' , T3'R3', and R3R3' with two types of HisBlO metal sites, the octahedral coordination geometry characteristic of T,, T,' and T,', and the pseudotetrahedral geometry of R,, R,' and R,'. The KNF model is free of oligomer symmetry constraints. Consequently, ignoring the possibility of subunit positional isomers, the simplest KNF model (Fig. 2A) predicts the existence of a minimum of six ligand-induced conformational states for the insulin hexamer: T,, T,R,, T4R2, T,R,, T,R4, T1R5, and R,. Due to the structural effects of the coil to helix transition of residues B1 to B8, it is reasonable to expect that adherence to the KNF model would give more than two geometries for the HisBlO metal sites. Fur- thermore, the 'H-NMR spectra of the hexamer mea-


61-810 HELIX

GLUB13 GLUB13-I

Fig. 8. Stereodiagram showing phenol bound to the phenolic pocket of the R, insulin hexamer and the interactions between the aromatic rings of phenol, HisB5, TyrB16, and TyrB26 (ball and stick structural elements). H bonds between the hydroxyl group of phenol and CysA6 and CysAll are shown as dashed lines. The backbone of a portion of the 8-chain helix (residues B1 to BIO is

sured as a function of ligand concentration would be expected to show the presence of more than three states.

is a symmetry- driven model constrained to two different, symmetry-conserved, quaternary conformations consisting of identical subunits. The T-state is defined as the tense, lower affinity state, and is usually the pre- dominant form when the protein is unligaged. The R-state is the relaxed, higher affhity state, and the thermodynamic consequence of ligand binding is to shift the distribution of states in favor of the high affinity form. We have previously shown that the MWC model is not suitable for describing the allosteric properties of the insulin h e ~ a m e r ~ - ~ for the reason that the symmetry constraints of the MWC model render it inappropriate for explaining negative cooperative ligand binding phen~mena.~. '~

The x-ray crystallographic evidence derived from the high resolution structures of the T,T,' , T3'R3', and R,R,' species shows that the HisBlO sites of the T,, T,' and T," units exhibit virtually identical octahedral geometries, while the sites of the R,, R,' and R," units have virtually identical pseudotetrahedral geometries. Because the R-state phenolic pockets are located on the monomer-monomer interfaces, subunit asymmetry in the dimeric units of T3OR3", and R3R3' give rise to three conformations of the phenolic pocket. The high resolution structures of T3OR3', and R,R,' species show that the pockets of an R," unit are different from those of R, and R,', while the R, and R,' units have almost identical

The MWC (or concerted)

shown together with the side chains of the three HisBlO residues, which make up one HisBlO zinc site (thin line structural elements). The positions and proximities of the rings of phenol, HisB5, Tyr B16, and TyrB26 are consistent with the upfield shifted resonances (Fig. 6) and NOE connectivities assigned to the C-4 proton of HisB5, and the 35 protons of TyrB16 and T ~ r B 2 6 . ~

conformations. The three phenol binding pockets of the SCN-- or C1-- stabilized T,R, structures",24 are occupied by water molecules. This crystallo- @aphic evidence is consistent with the symmetry relationships postulated for the SMB model shown in Figure 2B. (Indeed, the model was tailored to in- corporate this crystallographic evidence.)

In order to further test the models, the spectroscopic studies presented here are designed to deter- mine whether or not the R-state conformation pre- exists in solution in the absence of effectors for the phenolic pockets. Because the intensities and the energies of the Co(I1) d + d transitions are dependent on the coordination geometry of the Co(I1) cen- ter4,5,15,17,21,32 (Figs. 3 4 , Co(I1) substitution for Zn(I1) a t the His(B10) sites provides a useful method for studying the T- to R-state structural transition of the insulin hexamer. The octahedral Co(I1) T-state complex is characterized by a broad d + d envelope of low intensity (Am, = 495 nm, Em, = 45 M-' cm-l), whereas the tetrahedral Co(I1) R-state complexes are characterized by intense d + d profiles that are very sensitive to the structure of the fourth ligand and the nature of the coordinating atom. An- ions such as SCN- and N,- are known to stabilize a pseudotetrahedral Co(I1) center in the hexamer, both with and without phenolic ligands p r e ~ e n t . ~ , ~ . ' ~ High SCN- concentrations stabilize a T,R, species in both the Zn(I1) and Co(I1) insulin hexamers in s o l ~ t i o n . ' , ~ , ~ , ~ ~ Although the SCN- effect has been well characterized, the effects of halides, specifi- cally, C1- and I-, have received little attention.


Brzovic et al., were the first to report evidence indicating that C1- stabilizes Zn(I1) or Co(I1) R-state hexamers in solution in the absence of phenolic ligands. Our detailed spectral analysis of both the 'H NMR and Co(I1) UVNisible spectral data (Figs. 4-6) establish that traces of R-state-like structures exist in solutions containing high concentrations of halides. Spectral decomposition of the d + d transitions of the Co(I1)-substituted insulin hexamer shows that a t subsaturating ligand concentrations, the overall spectral envelope can be explained as a composite of just two spectra originating from an octahedral complex and a pseudotetrahedral complex (Fig. 5B). We assign these complexes as the metal sites of the T, and R, units of T, and T,R,, and we conclude that the anions are allosteric effectors that perturb the initial equilibrium between the T, and T,R, states. The ability of C1- ion to stabilize an R-state conformation is also evident in the 'H NMR spectrum (Fig. 6Be). Calculation of the relative area under the TyrB16 resonance using the standard lorentzian function shows that C1- anions stabilize about 13% of a T,R,-like structure. This demonstration of anion-stabilized R-states reaffirms our previous proposal that the T, to R, transition of the insulin hexamer is best portrayed by an SMB model (Fig. 2B) with concerted transitions involving the intermediate T,R, .6,7 The absence of x-ray crystal structures or solution spectroscopic evidence for more than three global hexamer conformations ar- gue against a KNF model for the insulin hexamer. Molecular dynamic studies (0. Olsen, personal com- munication) indicate the trimeric unit of the hexamer must undergo a concerted, coil to helix transition, not a sequential one.

Anion Binding and Negative Cooperativity in the Insulin Hexamer

Anion binding to the metal site is strongly linked via positive heterotropic interactions to ligand binding to the phenolic pockets, but there is no indication of allosteric interaction between the two His(B10) ~ i t e s . ~ , ~ , ~ The stabilization of the R-state by anions cannot overcome the thermodynamic energy barrier arising from the structural asymmetry between the two trimeric halves of the native insulin hexamer, consequently, an anion-mediated transition of T,R, to R, in wild-type human insulin does not occur. However, the mutation of GluB13 to Gln gives an insulin hexamer wherein the energies of the allosteric transitions are drastically altered.6,7,15 Roy et al.15 demonstrated that the metal-free hexamer can undergo conversion to the R, state by the binding of phenol. Brzovic et al., showed that SCN- can drive the Zn(1I)-substituted mutant hexamer to the Zn(I1)-R, state. Bloom et al.7 found that this mutation lowers the energy of the T3R3 to R, transition by 8 to 9 kcal/mole. Consequently, the energy barrier resulting from the intramolecular asymmetry be-

tween the two halves of the T,R, hexamer can be overcome either by the combined effects of monovalent anions and phenolic ligands, thus maximizing the heterotropic and the homotropic interactions, or by mutation of Glu(B13) to Gln.6.7 Therefore, in order to complete the T, + T,R, + R, transition (Fig. 2B), either the metal coordination changes must be combined with phenolic ligand binding to fulfill the energy requirement necessary to overcome the structural asymmetry of the T,R, hexamer, or intersub- unit contacts must be altered by mutation.

The SMB model (Fig. 2B), like the MWC model, predicts that a t subsaturating ligand concentrations, the fraction of sites occupied will be smaller than the fraction of sites that have undergone the conformational transition to the high affinity form. The work of Brzovic et al., has assigned the 'H- NMR resonance at 6.3 ppm to the 3,5 protons of TyrB16 in the R-state conformation. Due to the coil- to-helix conformation change, the ring of TyrBl6 is sandwiched between the rings of HisB5 and TyrB26 in the R-state (Fig. 8) but not in the T-state. The resulting ring current effects cause the large upfield shifts of the 3 3 protons of TyrB16 and TyrB26. When bound to the phenolic pocket, phenolic com- pounds make a van der Waals contact between the face of,;tghe phenolic ring and the C-4 ring proton of HisB5 ' (Fig. 8). The anisotropic ring current effects of this interaction cause the HisB5 C-4 ring proton to be strongly shifted upfield to 5.8 ppm in the complex with phenol (Fig. 7). The behavior of the TyrB26, TyrB16 and HisB5 resonances are inconsistent with the predictions of the KNF model.

The KNF model for allosteric interactions' (Fig. 2A) is based upon three primary assumptions: (1) In the absence of ligand, the protein exists in only one conformation, (2) only the subunit to which a ligand binds may change conformation, and (3) the ligand- induced conformational change in one subunit may affect the energies of other vacant subunits via protein-protein interactions thereby altering the ener- getics of the ligand-induced conformational transitions in those subunih2 In the case of the insulin hexamer, these assumptions imply that there should exist multiple conformational states (i.e., T,, T,R,, T,R2, .. R,) corresponding to the multiple states of ligation. Second, the KNF model cannot accommo- date the existence of vacant R-state ligand binding sites in the insulin hexamer, and a preexisting equilibrium between T and R forms is not allowed. These model constraints are not consistent with the 'H- NMR data shown for the phenol-induced T, to R, allosteric transition of the insulin hexamer (Fig. 7).

In the absence of other ligands, certain monovalent anions have been shown to stabilize the T,R, conformation of the insulin hexamer. As shown in Figures 6 and 7, the 'H-NMR spectrum of the Zn(II)-T, species predominates in the presence of C1- ion. However, a minor peak corresponding to


TyrB16, located at 6.52 ppm, indicates a small amount of T,R, species is present a t equilibrium. The chemical shift of this peak is characteristic of the position of the TyrBlS resonance in the T3R3 state stabilized by other monovalent anions (i.e., SCN-, I-, and N3J in the absence of phenolic ligands, and, therefore, represents the presence of “empty” phenolic binding sites. Addition of phenol results in a change in chemical shift of the TyrB16 resonance and an increase in peak area as the binding sites are filled and the concentration of insulin hexamer in the R-state is increased. Second, the data are consistent with a three state transition (T, Z T3R3 S R,)!,’ No other intermediate species are observed during the course of the titration.

The dynamics of ligand binding to the Zn(I1) insulin hexamer during the T, to T3R, transition dif- fers significantly from the rate of the protein conformational change. The behavior of the TyrB26 and TyrB16 resonances are indicative of slow exchange behavior for both the T, to T3R3 and the T3R, to R, conformational transitions. This observation is in agreement with transient kinetic studies of the T, to R, phenol-induced conformational transition.,’ Conversely, the line broadening and concentration- dependent chemical shift behavior of the HisB5 C-4 proton resonance indicate the exchange rate for ligand binding occurs on a much faster time scale than the protein conformational change. The HisB5 C-4 signal, which shifts dramatically upfield during the course of the titration (Fig. 7), occurs as a consequence of the particular juxtaposition of aromatic ring systems found in the R-state in the presence of bound phenolic ligand.4,6 The aromatic ring interactions require that the HisB5 side chain must move 8 to 10 in order to form part of the phenolic binding site. Therefore, if a rapid binding step precedes a slow protein conformational change, as would be predicted by application of the KNF model, then resonances unique to the R-state, including the HisB5 C-4 proton resonance, should exhibit slow exchange behavior. This is not the case. Although the overall T, to T3R, protein conformational change appears to be in the slow exchange regime, resonances per- turbed by the binding of the phenolic ligand exhibit considerably faster exchange behavior. These data are consistent with the rapid, reversible binding of phenol to preformed R-state binding sites following a slower redistribution of T- and R-state conformations. The KNF model is inconsistent with this behavior.

In conclusion, the allosteric transition of the insulin hexamer occurs via a mechanism that is consistent with the SMB model depicted in Figure 2, but cannot be explained by the KNF or MWC models. The transition is mediated by two types of binding phenomena: (a) binding of phenolic ligands to the R-state phenolic pockets and (b) binding of anionic ligands to the HisBlO metal sites of R, units. These

two binding sites are tightly linked by heterotropic interactions that stabilize the R-state conformation. The interdigitated head-to-tail arrangement of insulin subunits about the threefold axis of symmetry (Fig. l), together with asymmetry in the dimeric units greatly influence the allosteric properties of the hexamer (Fig. 2). This arrangement conveys nonidentical conformations to the trimeric units of the hexamer, and therefore gives two conformations to each class of ligand binding sites. These conformational differences are small in R, but large in T3R3. Consequently, the transition of T, to R6 via T3R3 exhibits mixed positive and negative cooperative behavior that is well described by the SMB

ACKNOWLEDGMENTS This work was supported in part by a gift from the

Novo Research Institute. We thank Curtis Bloom for helpful suggestions and for assistance in preparing some of the figures.

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Spectroscopic evidence for preexisting T- and R-state insulin hexamer conformations