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Tertiary Structure of Myohemerythrin at Low Resolution (Hemerythrin/Oxygen Transport/Sipunculan Worm/Protein Structure/X-Ray Diffraction) WAYNE A

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Tertiary Structure of Myohemerythrin at Low Resolution (Hemerythrin/Oxygen Transport/Sipunculan Worm/Protein Structure/X-Ray Diffraction) WAYNE A Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2160-2164, June 1975 Tertiary Structure of Myohemerythrin at Low Resolution (hemerythrin/oxygen transport/sipunculan worm/protein structure/x-ray diffraction) WAYNE A. HENDRICKSON*, GERALD L. KLIPPENSTEINt, AND KEITH B. WARD* *Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, D.C. 20375; and t Department of Biochemistry, University of New Hampshire, Durham, N.H. 03824 Communicated by I. M. Klotz, March 17, 1975 ABSTRACT X-ray diffraction studies have produced Many compounds seem to bind, but derivative crystals are a low resolution image and also located the iron atoms of a often prone to cracking and poor isomorphism. Nevertheless, monomeric hemerythrin from muscles of a sipunculan worm. These results reveal the course of the polypeptide five derivatives were found to be good enough at least for low- chain and some details of the active center. resolution phasing. X-ray diffraction data were measured by w-scans on a four- Oxygen transport in certain invertebrate animals is mediated circle diffractometer. CuKa radiation was used. Native data, by hemerythrin in erythrocytes of the coelomic fluid. He- including the Bijvoet pairs (hkl and hUl), were collected to merythrin usually occurs as an octamer of 108,000 molecular 2.8 A spacings. Derivative data were taken out to 3A spacings weight. It is a non-heme iron protein containing two iron from hkO, Okl, and hOl reflections and to 5.5 A spacings from atoms per subunit and it reversibly binds oxygen in the ratio Bijvoet pairs. Structure factor amplitudes were corrected for 1 02:2 Fe. Much study has been attended to the structural the effects of absorption (5), radiation damage (6), and the chemistry of this evolutionary alternative to hemoglobin as Lorentz-polarization factor. Native data were placed on an an oxygen carrier (1), particularly by Klotz and coworkers, absolute scale by statistical methods (6, 7), and derivative but many details remain obscure. New light can now be shed data were scaled to these, making appropriate allowance for on hemerythrin structure following the discovery by Klippen- the heavy-atom scattering (6). stein et al. (2) that the sipunculan worm Themiste (syn. Den- A (SF)2 Patterson map revealed the positions of a single drostomum) pyroides (3) contains a monomeric hemerythrin in site in the K3UO2F5 derivative. Then .F Fourier syntheses its retractor muscles as well as hemerythrin octamers in its based on phases derived therefrom permitted interpretation erythrocytes-a situation reminiscent of myoglobin and of the other derivatives while automatically referring them hemoglobin in mammals. Several properties of this myo- all to a common origin. The heavy-atom parameters were hemerythrin suggest that it bears close structural similarity refined separately for each derivative. Positions of the iron to the protomers of octameric hemerythrin (2, 4). atoms were derived from a |jFhklI - jF-j112 Patterson map Myohemerythrin from T. pyroides has been crystallized (4) (8) and then refined. Refinement results for the iron atoms and the first structural results from studies of these crystals and for the heavy-atom derivatives are shown in Table 1. are reported here. These results are mainly at low resolution, Phase information from the isomorphous replacement and but owing to a high helix content and recourse to chemical anomalous scattering data was cast in the ABCD formulation data, more molecular detail has been gleaned than is ordi- (9) to generate combined phase probability distributions. narily discernible at low resolution. By way of warning it Anomalous dispersion effects were also used to establish the should be noted that published interpretations of low-resolu- absolute enantiomorph (6). Fourier syntheses of the structure tion density maps have sometimes later been proved incorrect. were computed from centroid phases and figure-of-merit However, it seems unlikely that such mistakes are repeated weighted structure amplitudes (10, 11). Information from here. The quality of this map and the consistency of the three derivatives was used in computing a first Fourier map. model with independent chemical data argue for the basic The 490 terms included at 5.5 A resolution had a mean correctness of the rather detailed molecular interpretation figure-of-merit, mn, of 0.83. A second map based on all five given here. derivatives had mn = 0.89. Finally, the phases were improved Myohemerythrin is a relatively small protein of 118 amino- by a solvent constraint refinement procedure (manuscript acid residues and molecular weight 13,900. This facilitates in preparation) to yield a third map with mn = 0.92. its crystallographic analysis. In turn, the knowledge of this structure should simplify the analysis of octameric hemeryth- Interpretation of Fourier maps rins. In any event, further studies on myohemerythrin should The interpretation of even the first Fourier synthesis of be an avenue for gaining a detailed understanding of reversible myohemerythrin was uncommonly straightforward. Apart oxygenation in this fascinating class of proteins. from one ambiguity, the bounds of a single contiguous mole- cule were readily apparent. Most salient among features in X-ray analys the density maps are four rather parallel dense rods, each Crystals of metazide myohemerythrin were grown as pre- 30-40 A long, and an especially dense spheroidal mass, 7-10 A viously described (4) and then transferred to a stabilizing in diameter, which they embrace. A more tortuous stretch medium of 80% saturated ammonium sulfate buffered to of density is appended to one side of these features. The pH 6.7. These crystals are in space group P2,2121 and have ambiguity arose in determining the mode of attachment of unit cell dimensions of a = 41.58 A; b = 80.03 A; and c = this arm to the main body of density. An initial interpretation 37.78 X. Derivatives were prepared by soaking native crystals of this attachment has been abandoned in favor of an alter- in stabilizing media containing heavy-atom compounds. native which is more reasonable on several grounds. It has 2160 Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 72 (1976) Structure of Myohemerythrin 2161 TABIE 1. Parameters from heavy atom refinements Derivative Aa/a Ab/b Ac/c Atom q x y z B R Q P Hg(CN)2 1.73% 0.57% 0.03% Hgl 0.84 0.475 0.746 0.273 18 ,2 0.51 0.206 1.44 Hg2 0.62 0.407 0.807 0.331 10 KAu(CN)- 0.50 0.09 0.30 Aul 0.42 0.471 0.746 0.290 11 0.49 0.070 1.68 Au2 0.28 0.497 0.728 0.222 8 K3UO2F5 -0.55 -0.05 -0.26 U1 0.47 0.266 0.451 0.158 16 0.51 0.056 1.59 K2Pt(CN)4 -1.88 0.60 -0.71 Ptl 0.93 0.840 0.519 0.176 11 0.47 0.226 1.92 Pt2 0.49 0.444 0.416 0.045 13 Pt3 0.42 0.139 0.773 0.269 9 K1PtCI4 1.03 0.10 -0.34 Ptll 0.85 0.521 0.485 0.630 25 0.44 0.127 1.65 Ptl2 0.44 0.579 0.554 0.076 54 Native anomalous Fel 0.80 0.539 0.548 0.323 5 0.40 Fe2 0.92 0.479 0.561 0.379 4 Structure factor contributions from the heavy atoms were calculated as fH = Zqif(s) exp (- Bis2) exp 27ri(h xi + ky1 + lzi) where s = sin O/A. Scattering factors, f(s), were taken asfo + Af' from tabulations for neutral atoms. The adjustable parameters are q, the fractional occupancy; x, y, and z, the atomic positions in fractions of a cell edge; and B, the isotropic "temperature" factor. The sum is over all sites in the unit cell. For centric reflections, observed amplitudes were taken as AF = FH - Fp, where FH is the structure amplitude for the heavy-atom derivative and Fp is that for the native protein. Probable sign-reversal reflections (6) were excluded from the refinements. For general reflections, observed amplitudes were estimated by Matthews' coefficient (38) formed with a relative weighting for Bijvoet differences chosen such as to equate the averages of these coefficients with averages of AF in the centric zones. These refinements were based on the 668 centric reflections with spacings greater than 3 A and the 300 general reflections to 5.5 A. The number of sign-reversal exclusions ranged from 17 to 153. The refinement of iron atom positions was based on the structure factor contributions from anomalous scattering in the native protein. Observed structure amplitudes were taken as IFk1 - Fai-i/2 and, accordingly, scattering factors were assumed to be f(s) = Af" = 3.45e, independent of s. These observations systematically underestimate the true anomalous scattering amplitude by about a factor cos ' where ip is the phase difference between the anomalous scattering vector and the total "real atom" structure factor. However, the exclusion of weak Bijvoet differences tends to eliminate reflections for which cos 4, is small. Thus, only reflections for which Fhkk - FhkI exceeded one sigma for the difference were included in the refinement. A total of 1504 of the 2627 general reflections at 2.8 A resolution qualified. Nonetheless the effect of cos 4, is not fully compensated so occupancies are somewhat underestimated here. In all cases, the R-value cited is R = ZjFo - FJ1/2Fo where Fo and F, are observed and calculated magnitudes of structure factors against which the refinement was made. Sums are taken over all data included in the refinement. The ratio Q = ((AF)2)/(Fp2) is a measure of the scattering power of the heavy atoms-relative to that of the protein in an isomorphous derivative.
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