Spectroscopic Signatures of the T to R Conformational Transition in the Insulin Hexamer ”

The cobalt(I1)-substituted human insulin hexamer has been shown to undergo the phenol-induced T6 to Re structural transition in solution. The accompanying octahedral to tetrahedral change in ligand field geometry of the cobalt ions results in dramatic changes in the visible region of the electronic spectrum and thus represents a useful spectroscopic method for studying the T to R transition. Changes in the Co2+ spectral envelope show that the aqua ligand associated with each tetrahedral Co2+ center can be replaced by SCN-, CN-, OCN-, N;, C1-, and NO;. “F NMR experiments show that the binding of m-trifluorocresol stabilizes the Re state of zinc insulin. The chemical shift and line broadening of the CFs singlet, which occur due to binding, provide a useful probe of the “6 to Re transition. Due to the appearance of new resonances in the aromatic region, the 500 MHz ‘H NMR spectrum of the phenol-induced Re hexamer is readily distinguishable from that of the T6 form. ‘H NMR studies show that phenol induces the T6 to R6 transition, both in the (GlnB13)6(Zn2+)2 hexamer  and  in  the metal-free GlnB13 species; we conclude that metal binding is not a prerequisite for formation of the R state in this mutant.

** TO whom all correspondence should be addressed.
ignated &).I The Tg, T3R3, and & designations (17) appear to be gaining wide acceptance (13). Interconversion of the T and R conformations (the T to R transition) involves an extended chain to helix transition of residues B1-B8 ( Fig. 1) in which some residues move by as much as 20-25 A (12,13).
It is unclear what the relevance of either structure is to the biologically active form of insulin (the insulin monomer).
When subjected to lyotropic anions such as SCN-, I-, Br-, or C1-(ll), three of the six subunits of the crystalline zinc insulin hexamer undergo the T to R conformation change to give T3R3. Crystallization in the presence of phenol gives an & hexamer in which all six subunits have undergone the T to R conformation change (13). This conformational transition creates six essentially identical, well defined pockets within the hexamer that bind phenol via hydrophobic and Hbonding interactions. The available spectroscopic evidence from 'H NMR (18,19) and circular dichroism (20, 21) and the rapid kinetic studies of Kaarsholm et al. (17) indicate that the conformational states found in the crystal can be induced in solution by the binding of lyotropic anions or by phenol. The x-ray crystallographic studies have shown that, in the crystal, the T6 zinc hexamer (Fig. lA) incorporates two Zn2+, each coordinated in an octahedral arrangement by three his-tidy1 B10 nitrogens and three water molecules. In the & form ( Fig. lB), there are two identical zinc binding sites in which each Zn2+ is coordinated in a tetrahedral arrangement by three B10 histidyl nitrogens and a water molecule. If the conformational behavior of the cobalt(I1)-substituted insulin hexamer (In)6(Co2'),, parallels that of the zinc hexamer, (In)6(Zn2+)2, then the different ligand fields experienced by the cobalt(I1) ion in the T6 and R6 conformations should be manifested as distinct electronic spectral signatures of the two forms (cf. the behavior of carbonic anhydrase, carboxypeptidase, and alcohol dehydrogenase (22)).
In this communication, we establish that the T6 to R, conformational transition occurs in the (In)6(CoZ')2 hexamer and induces a change in the ligand field about Co2+ from octahedral to tetrahedral. Via 'H and "F NMR, UV-visible, CD spectroscopy, and rapid kinetics, we have undertaken studies to investigate the nature of the T6 to R, conformational transition and the phenol binding process. We report here our preliminary findings from these studies and show that these spectroscopic tools can be used to quantitate the kinetics and thermodynamics of the T6 to & transition in nated as (In)6(MZ+)2 where M2+ is either Zn'" or Co2+. The GluB13"t The abbreviations used are: human insulin hexamers are desig-Gln mutant hexamer is designated as (GlnB13)6, the metal-free hexamer; and as (GlnB13)6(Zn2+)2, the zinc-substituted hexamer. The crystalline hexamers are designated as follows: T , 2Zn insulin; TBR3, 4Zn insulin; &, phenol-induced hexamer; ppm, parts per million.

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This is an Open Access article under the CC BY license. solution and that metal coordination is not a prerequisite for the conformation change.

Methods
Metal-free insulin stock solutions containing e0.002 mol of Zn2+ per insulin monomer were prepared as described previously (6,17,19,23). The metal ions of choice were added as required just prior to use.
UV-oisible Spectra, CD Spectra, and Kinetic Studies-UV-visible spectra were collected on a Hewlett-Packard 8450A UV-visible spectrophotometer. CD spectra were recorded on a Jobin-Yvon Dichrograph Mark V. Single wavelength, rapid kinetic measurements and the kinetic analyses were made as described previously (23,24). All experiments were carried out in 50 m M Tris-HCI, pH 8.0. N M R Spectra-Samples for NMR spectroscopy were typically 1-2 mM insulin. IH NMR spectra were recorded a t 25 "C on a GN-500 spectrometer equipped with a Nicolet 1280 computer. Chemical shifts are reported in parts/million relative to the methyl resonance of 2,2dimethyl-2-silapentane 5-suIfonate-2,2,3,3-d4. "F NMR spectra were recorded on a Nicolet NT300 NMR spectrometer. A capillary tube containing 60 mM rn-trifluorocresol as reference standard was inserted into the sample tubes. The temperature of the sample probe was thermostated at 26 "C. A DzO field frequency lock was used for both "F and 'H NMR measurements.

RESULTS
The visible electronic absorption spectrum of (In)6(Co2+)2 is shown in Fig. 2 A , a). The broad and weakly intense absorption band centered on 495 nm is typical of d 3 d transitions observed in octahedral cobalt(I1) complexes (25). Fig. 2 A , be, shows the effects of increasing concentrations of phenol on the 400-700 nm region of the absorption spectrum in the presence of Cl-. The appearance of intense bands (X, = 580 nm, c, = 1000 M" cm", and hlmax = 558 nm, c2 = 700 M" cm" (shoulder)) are consistent with the d 4 d transitions expected for a tetrahedral cobalt(lI)-Cl-complex (22, 25) and graphically illustrate that phenol promotes the conversion of the cobalt metal centers from octahedral to tetrahedral coordination geometry. Furthermore, marked changes occur in the CD spectrum of (In)6(Coz+)2 upon the addition of phenol. Fig.  2B, a, shows that the CD spectrum of (In)6(Co2')2 exhibits a CD band centered on 500 nm. Fig. 2B, b, (Fig. 3A, inset) shows the occurrence of a saturation phenomenon with KOs -2 mM.
The spectra in Fig. 3B show that phenol induces the Tg to I & transition both in the mutant (GlnB13)6(Zn2+)2 hexamer (compare spectra a and b and in the metal-free GlnB13 species spectrum c ) . Comparison of spectra b and c strongly suggests that the metal-free species is a hexamer with the & conformation.
To further investigate the binding process, we have utilized "F NMR spectroscopy to qualitatively study the interaction of rn-trifluorocresol with zinc-insulin by observing the chemical shift of the CF3 singlet. The "F NMR spectra (Fig. 4),

Insulin Conformational Transitions
measured with varying concentrations of m-trifluorocresol show that in the presence of (In),(Zn'+),, the CF3 singlet is broadened and shifts downfield. The observed singlet indicates that rn-trifluorocresol is in rapid to intermediate exchange (relative to the NMR time scale) between the insulinbound environment and that of the solution, The line broadening, which usually occurs when a small molecule binds to a macromolecule, is interpreted as indicative of intimate contact between the fluorine atoms of m-trifluorocresol and the phenol binding site of the zinc-insulin hexamer. Spectra a-c show that in the presence of (In)6(Zn'+)2 the chemical shift of the CF, singlet is dependent upon the m-trifluorocresol concentration. The CF3 resonance is shifted upfield as the rntrifluorocresol concentration is increased from 0.5 to 5.0 mM, reflecting the influence of the increasing proportion of unbound rn-trifluorocresol on the averaged chemical shift. Spectrum d shows the effect of incorporating 17 mM &-phenol into a sample of 2 mM zinc-insulin with 2.5 mM m-trifluorocresol. Comparison of spectra b and d indicates that phenol displaces m-trifluorocresol from the binding sites of the insulin hexamer and that the position of the CF, resonance shifts toward that of the unbound ligand with an accompanying narrowing of the linewidth. Control experiments performed in the absence of insulin show that the chemical shift of the m-trifluorocresol CFs resonance is essentially invariant over the concentration range 0.5 to 5.0 mM and also invariant to the presence of phenol.

DISCUSSION
In the hemoglobins of higher organisms, the binding of dioxygen forces a change in the coordination geometry of the heme iron from 5-to 6-~oordinate. This reduces the Fe-Pj bond distances by -0.2 A, alloying the iron to move -0.6 A into the heme plane. The 0.6-A motion triggers a conformation change that alters subunit interfaces, moves heme groups on the /3 subunits closer together by as much as 6 A and increases the affinity for dioxygen by 2 orders of magnitude (26)(27)(28). The single-crystal x-ray diffraction studies of Derewenda et al. (13) show that the phenol-mediated Tfi to €& transition of (In)6(Zn'+)Z involves a gross molecular rearrangtment of all six insulin B chains in which B1 moves 20-25 A. This allosteric transition changes the topography of the hexamer surface, creates six binding sites for phenol and dictates a new metal coordination geometry at the HisBlO sites (Fig. 1).
Our spectroscopic and kinetic studies establish that phenol or m-trifluorocresol mediate large changes in the conformations of the (GlnB13)6(Zn'+)~, (In)6(Zn2+)2, (In)6(Co2+)2, and metal-free (GlnB13)G hexamers in solution. Since the absorbance and CD electronic spectral changes induced by phenol (Fig. 2) are only reasonably explained as arising from the transformation of the Co'+ ligand field from octahedral to tetrahedral, we conclude that the phenol-mediated spectroscopic signatures identified in these studies have their origins in a conformational change that corresponds to an allosteric transition between the crystallographically identified T6 and states. Using the same spectroscopic criteria, it appears that SCN-and OCN-also induce T to R transitions in the Zn'+-and Co'+-substituted hexamers (data not shown).
The GluB13 carboxylates of (In)6(Zn'+)2 have been shown to form a cage that binds Ca'+ (19,(29)(30)(31)(32). In the absence of Ca2+ or other divalent metal ions, Coulombic charge repulsions between the six B13 Glu carboxylates destabilize the hexamer (19,30,33). The GlnB13 mutant (34) lacks this Ca'+ site and was designed to enhance the stability of the hexamer and thus slow the release of insulin following subcutaneous injection in diabetes therapy.' Osmotic pressure molecular weight studies (data not shown) show that the metal-free GlnB13 mutant is predominantly a molecular weight 36,000 species at pH 7.5 and 1-10 mg/ml, while in the same concentration range, the molecular weight of metal-free human insulin is variable and less than 36,000. The data in Fig. 3 indicate that phenol induces a conformational change in the metal-free (GlnB13)e that is highly similar to that induced in (In)dZn'+)~ and in (GlnB13)6(Zn2')z; hence, metal binding is not essential for the allosteric transition. Circular dichroism studies on the GlnB13 mutant (35) have led to similar conclusions. Under the same conditions of pH and insulin concentration, we find no evidence that phenol induces an €& state in metal-free human insulin. The rate of the T to R transition is 6-8 orders of magnitude slower than that expected for a simple coil-to-helix peptide transition in solution (36).
In the T state, the HisBlO zinc sites are positioned at the bottom of shallow clefts at opposite ends of the cylindrically shaped hexamer, Fig. 1B. The three water molecules coordinated to each octahedral Zn2+ ion extend out into the cleft and can be readily replaced by tridentate chelators (17,23,30,31). In the R state, the HisBlO sites are buried, and the only obviou! access to the tetrahedrally coordinated Zn'+ ion is via an 8-A-long tunnel that is too narrow to accommodate large tridentate chelators (12,17). Our studies (Fig. 2 A ) show that in the & form of (In)6(Co2+)2, this site is accessible to small anions such as CN-, OCN-, N;, SCN-, NO,, C1-, and that these anions can replace the coordinated HzO with an accompanying change in the UV-visible electronic spectrum. Phenol binding stabilizes all six of the insulin subunits in the R conformation, giving an €& structure, whereas in the crystalline state, the transition induced by lyotropic anions converts only three of the six insulin subunits to the R state, giving a T3R3 structure. The anion-induced T3R3 crystalline hexamer may be constrained to the T3R3 state by crystal lattice forces (10,12). It is not known whether or not the T3R3 state is a stable species in solution. Four positions and three types of zinc sites are identified in the T3R3 crystal structure. Some of these sites involve both HisB5 and HisB10. However, the total Zn'+ occupancy was calculated to be 2.67 (12). In the phenol-induced €& structure, there are only two identical, high affinity tetrahedral Zn2+ sites (Fig. lB), each is made up of three HisBlO imidazole rings and one water molecule. Phenol is bound via a large number of van der Waals contacts between the ring and various side chain atoms of the A and B chains and via two hydrogen bonds between the phenolic hydroxyl and the A chain backbone, one to the carbonyl oxygen of CysA6, the other to the amide N-H of CysAll (13). The side chains of both HisBS and HisBlO (from adjacent subunits) come close to the same phenol ring. The disappearance of the HisB5 and B10 C-2 proton NMR signals (Fig. 3) probably is due to anisotropic ring current effects from the phenol molecules which shift these signals upfield. Either the C-2 proton signals are located under the aromatic envelope (6.5 to 7.5 ppm) or they appear as new signals between 5.0 and 6.5 ppm. The resonances located between 5.0 and 6.5 ppm almost certainly are due to ring current effects either from phenol or from new interactions involving the aromatic side chains of insulin. The spectrophotometric titration of (In)6(Co2+)z with phenol gives rise to an isotherm which may be approximated by two hyperbolic functions. This suggests that the binding of phenol is a negative cooperative process which would presumably arise from allosteric interactions in the T 6 to It6 transition.

Insulin Conform1
The high resolution x-ray structures of the T6, T3R3, and R6 states, show that the phenol-and anion-mediated conformation changes of the insulin hexamer are interesting examples of an allosteric transition. Because there are only a few examples of allosteric systems where the details of the changes in three-dimensional structure are well defined, this allosteric transition seems worthy of further investigation. The spectroscopic probes described in this study make it possible to carry out detailed thermodynamic and kinetic investigations to determine the mechanism of the transition.