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Inorg. Chem. 2000, 39, 5422-5423
Arsenic(III)-Cysteine Interactions Stabilize Three-Helix Bundles in Aqueous Solution Brian T. Farrer, Craig P. McClure, James E. Penner-Hahn, and Vincent L. Pecoraro* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109
ReceiVed September 7, 2000 The area of de novo peptide design has been dominated by the formation of R-helical bundles.1 R-Helices can aggregate in solution to form two-, three-, and four-helix bundles.2-4 The determining factors defining the preferred aggregation state are still being clarified, although subtle changes in the position and identity of hydrophobic residues play a role.5 The presence of interhelical salt bridges can also be important by employing negative design strategies.2 Since these interactions are relatively weak, the difference in energies between two-, three-, and fourhelix bundles are small. Metal-ligand bonds are many times stronger than noncovalent interactions that stabilize protein structure such as hydrophobic or cation-π interactions, salt bridges, and hydrogen bonds.6 Only covalent interactions such as disulfide bonds are comparable in energy to metal-ligand bonds. Disulfide bonds, however, are binary and connect only two strands or subunits per bond, while metal sites are able to cluster several units at a single site. De novo three-helix bundle stabilization by ruthenium(II) has been achieved using unnatural, bulky amino acids containing a strongly chelating 2,2′-dipyridyl moiety capping the bundle.7 Arsenic(III) is found primarily with trigonal-pyramidal coordination geometry, with expansion to tetrahedral geometry found in some cases.8 Despite the nearly 18 million cases of arsenic poisoning resulting from contaminated drinking water, the chemistry of As(III) with biomolecules has scarcely been explored.9 As(III) has been shown to interact with glutathione to produce a 3:1 glutathione:arsenic complex through thiolate sulfur atoms,10 and cysteine reacts with arsenite to produce an As(Cys)3 complex with coordinated thiolates.11 EXAFS spectroscopy indicates a 3-coordinate cysteine environment for arsenic bound to the arsenic regulatory protein, ArsC.12 The preference of arsenic (1) (a) DeGrado, W. F.; Summa, C. M.; Pavone, V.; Nastri, F.; Lombardi, A. Annu. ReV. Biochem. 1999, 68, 779-819. (b) Hodges, R. S. Biochem. Cell Biol. 1996, 74, 133-154. (c) Shifman, J. M.; Moser, C. C.; Kalsbeck, W. A.; Bocian, D. F.; Dutton, P. L. Biochemistry 1998, 37, 1681516827. (d) Gibney, B. R. J. Am. Chem. Soc. 1999, 121, 4952-4960. (2) Dieckmann, G. R.; McRorie, D. K.; Lear, J. D.; Sharp, K. A.; DeGrado, W. F.; Pecoraro, V. L. J. Mol. Biol. 1998, 280, 897-912. (3) Johansson, J. S.; Gibney, B. R.; Skalicky, J. J.; Wand, A. J.; Dutton, P. L. J. Am. Chem. Soc. 1998, 120, 3881-3886. (4) Skalicky, J. J.; Gibney, B. R.; Rabanal, F.; Urbauer, R. J. B.; Dutton, P. L.; Wand, A. J. J. Am. Chem. Soc. 1999, 121, 4941-4951. (5) (a) Cohen, C.; Parry, D. A. D. Proteins 1990, 7, 1-15. (b) Beasley, J. R.; Hecht, M. H. J. Biol. Chem. 1997, 272, 2031-2034. (6) (a) Matthews, B. W. Annu. ReV. Biochem. 1993, 62, 139-160. (b) Fersht, A. R.; Serrano, L. Curr. Opin. Struct. Biol. 1993, 3, 75-83. (7) (a) Ghadiri, M. R.; Soares, C.; Choi, C. J. Am. Chem. Soc. 1992, 114, 825-831. (b) Case, M. A.; Ghadiri, M. R.; Mutz, M. W.; McLendon, G. L. Chirality 1998, 10, 35-40. (8) Dhubhghaill, O. M. N.; Sadler, P. J. Struct. Bonding 1991, 78, 129190. (9) (a) Lepkowski, W. Chem. Eng. News 1998, 76 (46), 27-29. (b) Aposhian, H. V. Annu. ReV. Pharmacol. Toxicol. 1997, 37, 397-419. (c) Acharyya, S. K.; Chakraborty, P.; Lahiri, S.; Raymahashay, B. C.; Guha, S.; Bhowmik, A. Nature 1999, 401, 545. (d) Nickson, R.; McArthur, J.; Burgess, W.; Ahmed, K. M.; Ravenscroft, P.; Rahman, M. Nature 1998, 395, 338. (10) Server, S.; Charalambidis, Y.; Sotiropoulos, D.; Ioannou, P. Phosphorus, Sulfur Silicon Relat. Elem. 1995, 105, 109-116. (11) Voegtin, C.; Johnson, J. M. J. Biol. Chem 1930, 89, 27.
Scheme 1
Table 1. Peptide Aggregation State and Metal-Sulfur Bond Distances for Apo, Hg(II), and As(III) TRI Family Peptides L16C and L12C pH 8.5 5.5
8.5 5.5 a
Apo aggregation M-S distance aggregation M-S distance aggregation M-S distance aggregation M-S distance
L16C three two L12C three two
Hg(II)a
As(III)
three 2.39 Å two 2.30 Å
three 2.25 Å three 2.25 Å
two 2.32 Å two 2.32 Å
three 2.24 Å three 2.24 Å
From ref 13.
for trigonal thiolate coordination environments makes the As(III)-cysteine interaction an excellent candidate for stabilization of three-helix bundles. The two peptides used for this study are members of the TRI family of peptides shown in Scheme 1. These peptides are entirely composed of naturally occurring amino acids. The aggregation of these peptides is pH dependent, with two-helix bundles forming at low pH (pH e 5.5) and three-helix bundles at high pH (pH g 7.5).2 Substitution of a leucine residue for cysteine at position 12 (L12C) or 16 (L16C), introduces a thiolate for metal binding. Studies with Hg(II) demonstrated the inequivalence of the 12 and 16 sites.2,13 At low pH, where metal coordination number and peptide aggregation state work in concert, stable 2-coordinate Hg(II) complexes in a two-helix bundle were formed. However, at high pH, the metal and peptide preferences conflict; Hg(II) prefers a coordination number of two, while peptide association favors three-helix bundles. For L12C, the Hg(II) preference dominates, giving two-helix bundles and 2-coordinate Hg(II), while for L16C, the peptide preference dominates, giving threehelix bundles and 3-coordinate Hg(II) (Table 1). Addition of a 100-fold excess of NaAsO2 to L16C in potassium phosphate buffer (pH 8.5) and subsequent purification by HPLC on a C18 reversed-phase preparative column at pH 2 afforded a product which gave a mass of approximately 10300 by both electrospray and MALDI mass spectrometries (Figure 1).14 This mass corresponds to an As(L16C)3 product (MW ) 10305). The product showed significant R-helical content (85 ( 5%) by circular dichroism. Several aspects of this synthesis indicated that (12) Liu, J.; Rosen, B. P. J. Biol. Chem. 1997, 272, 21084-21089. (13) Dieckmann, G. R.; McRorie, D. K.; Tierney, D. L.; Utschig, L. M.; Singer, C. P.; O’Halloran, T. V.; Penner-Hahn, J. E.; DeGrado, W. F.; Pecoraro, V. L. J. Am. Chem. Soc. 1997, 119, 6195-6196. (14) MALDI and electrospray mass spectrometries were performed by the Protein and Carbohydrate Structure Facility, University of Michigan.
10.1021/ic0010149 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/03/2000
Communications
Figure 1. Mass spectra of As(L16C)3 mixing 100 µM L16C and 10 mM NaAsO2 in 20 mM potassium phosphate (pH 8.5) after isolation using a C18 reversed-phase preparative column. (A) Electrospray ionization mass spectrum. Peaks correspond to degree of ionization (e.g., A10 is the species bearing a +10 charge). (B) MALDI-TOF of the same sample as part A.
Figure 2. EXAFS data for (A) As(L12C)3 and (B) As(L16C)3 both at pH 8.5. Identical EXAFS data are found at pH 5.5. Solid lines are data; dashed lines are fits using three sulfur neighbors.
arsenic stabilizes three-helix bundles. First, the synthesis was carried out with excess arsenite, a condition that favors formation of lower aggregation states. Second, although the addition of arsenite to the peptide occurred at pH 8.5, a condition where the peptide is stable as a three-helix bundle, the purification of the peptide was carried out under acidic conditions (0.1% trifloroacetic acid, pH 2) where the peptide prefers to be a two-helix bundle in the absence of arsenic. Finally, the three-helix bundle was stable to the conditions of mass spectrometry, with no free peptide observed in the electrospray mass spectrum. The oxidation state and the atoms in the first coordination sphere of the arsenic were determined by X-ray absorption spectroscopy.15 Comparison of the arsenic K-edge energies of the arsenic peptides with those for aqueous NaAsO2, As(glutathione)3, and As(V)O43- verified an As(III) oxidation state. EXAFS data (Figure 2) supported an As-S bond length of 2.25 Å,16 in agreement with both published trithiol ligation distances,12,17 as well as EXAFS data for As(glutathione)3. NaAsO2 was added to Tri L16C at pH 5.5. The product showed the same HPLC retention time found when the reaction was (15) XAS data measured at SSRL line 9-3 using a Si(220) double crystal monochromator and a Rh-coated Ni mirror for harmonic rejection. Arsenic fluorescence measured with a 30-element Ge solid-state detector. X-ray energies calibrated using As foil as an internal standard, with the first inflection point defined as 11867 eV. Data reduction followed standard procedures. Clark-Baldwin, K.; Tierney, D. L.; Gouindawamy, N.; Gruff, E. S.; Kim, C.; Berg, J. M.; Koch, S. A.; Penner-Hahn, J. E. J. Am. Chem. Soc. 1998, 120, 8401-8409. (16) EXAFS data fit using amplitude and phase parameters calculated with FEFF 6.01, and calibrated using EXAFS data for As(glutathione)3 (AsS) and As(OH)3 (As-O). The calibrated scale factor (1.0) and ‚Eo (-10 eV) were held constant during fits to peptide EXAFS data. (a) Rehr, J. J.; Deleon, J. M.; Zabinsky, S. I.; Albers, R. C. J. Am. Chem. Soc. 1991, 113, 5135-5140. (b) Rehr, J. J.; Albers, R. C.; Zabinsky, S. I. Phys. ReV. Lett. 1992, 69, 3397-3400.
Inorganic Chemistry, Vol. 39, No. 24, 2000 5423 carried out at pH 8.5, and a three-helix bundle was observed by electrospray mass spectrometry. Because the arsenic-free peptide is a two-helix bundle at pH 5.5, this result suggests that formation of As(L16C)3 is not only kinetically but also thermodynamically favored at low pH. Addition of excess NaAsO2 to L12C at high or low pH gave a three-helix bundle with a molecular weight identical to that for the aggregate made with L16C.18 EXAFS data confirm trigonal As-S ligation similar to that seen with L16C. In contrast to Tri L16C, L12C is a peptide that does not coordinate Hg(II) in a trigonal fashion under any conditions, demonstrating that L12C is not as suited for trigonal ligation as is L16C. Modeling studies suggest that unfavorable rotameric forms of the cysteine are needed in order to form a trigonal binding site in L12C.2 The ability of As(III) to stabilize the three-helix bundle of L12C attests to the propensity of As(III) to stabilize three-helix bundles by forming trigonal-pyramidal thiolate structures. By monitoring As(L16C)3 by HPLC, the stability of the arsenic(III) complex under a variety of conditions can be tested. The three-helix bundle was stable for 12 h in a wide range of pH’s and in the presence of 1 M KCl at pH 5.5 and 8.5. HgCl2 was observed by HPLC to displace the As(III) in the presence of stoichiometric amounts of AsIII(L16C)3 and HgCl2 at pH 8.5 (Figure S3, Supporting Information). In the presence of excess As(III), Hg(II) did not displace As(III). The product of Hg(II) displacement of As(III) from As(L16C)3 is Hg(L16C)2, a two-helix bundle. This result indicates that the structure of the peptide is dependent on the relative concentration of arsenic and mercury in solution, giving rise to a molecular switch sensitive to the presence of specific types of heavy metals. In summary, these results demonstrate that As(III) is able to distort polypeptide structure in order to satisfy its desire to form trigonal-pyramidal thiolate coordination. First, As(III) is trigonally ligated by L12C at pH 8.5, presumably overcoming an energetic barrier and forming unfavorable rotameric forms of the cysteine in position 12 that prohibits trigonal ligation to Hg(II).13 Second, As(III) is trigonally ligated by both L12C and L16C at pH 5.5, overcoming the favored two-helix bundle conformation of the peptides in the absence of arsenic. These results also indicate the flexibility of L16C, showing that it is able to bind not only to Hg(II) forming a trigonal-planar, negatively charged site but also to As(III) forming a trigonal-pyramidal, neutral site. Furthermore, these results demonstrate that arsenic can cause structural distortion and aggregation in biopolymers, an action which may be involved in the mechanism of arsenic toxicity. Acknowledgment. B.T.F. and V.L.P. thank Prof. Barry Rosen and Prof. William DeGrado for helpful discussions. B.T.F. thanks NIEHS for NRSA Grant 1 F32 ES05888-01, J.E.P.-H. thanks the NIH for Grant GM 38047, and CPM thanks Michigan ChemistryBiology Interface Training Program, NIH Grant GM 08597. X-ray absorption data were measured at SSRL, which is operated by the DOE, Office of Basic Energy Sciences, with additional support from the NIH, National Center for Research Resources and the DOE Office of Biological and Environmental Research. Supporting Information Available: Chromatograms of As(L16C)3 and As(L12C)3, HPLC conditions, competition studies between As(III) and Hg(II), electrospray mass spectra of As(L12C)3, and EXAFS data of all model compounds and peptide complexes. This material is available free of charge via the Internet at http://pubs.acs.org.
IC0010149 (17) Pappalardo, G. C.; Chakrovorty, R.; Irgolic, K. J.; Meyers, E. A. Acta Crystallogr. 1983, C39, 1618-1620. (18) The electrospray of As(L12C)3 prepared at pH 5.0 showed the presence of free L12C (m ) 3411) along with the major envelope for As(L12C)3, indicating decreased stability of As(L12C)3 compared to As (L16C)3.
