Crystal structure of sickle-cell deoxyhemoglobin at 5 Å resolution

Wishner, B.C.; Ward, Keith B.; Lattman, Eaton E.; Love, Warner E.

doi:10.1016/s0022-2836(75)80108-2

Cited by 336 publications

(137 citation statements)

References 20 publications

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“…We have calculated structure factors and crystallographic R factors for the molecular models resulting from both the high-and low-resolution searches (Table 4). These numbers compare favorably with the R factors of other structures at similar stages of their solution (12)(13)(14)(15).…”

Section: Methodssupporting

confidence: 57%

Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5-A resolution.

Navia¹,

Segal²,

Padlan³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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An electron-density map of the mouse galactan-binding immunoglobulin 1539 (IgA2,K) Fab has been calculated to a resolution of 4.5 A by the method of heavy atom isomorphous replacement with four derivatives. The map has been interpreted with the aid of a com uter program which systematically searched for the best fit between the electrondensity map and the known coordinates of individual immunoglobulin domains. The quaternary structure of J539 Fab at this resolution appears similar to that of another mouse immunoglobulin, IgA2,K Fab, McPC603. The model coordinates for J539 Fab should allow us to proceed directly to a high-resolution structure determination without further heavy atom isomorphous replacement.The crystal structures of three immunoglobulin Fabs have been reported (1-3). One of these, the mouse myeloma protein McPC603, belongs to a class of immunoglobulins that bind specifically to phosphocholine. In this paper, we present the results of.a structural analysis at 4.5-A resolution of a second mouse Fab, the galactan-binding immunoglobulin J539. A molecular model has been constructed with the aid of a computer search procedure used previously in the interpretation of the electron-density map of an intact immunoglobulin (4). The model allows us to describe the quaternary structural interactions between the constituent domains and domain pairs of the J539 Fab and reveals a general similarity to McPC603. MATERIALS AND METHODSThe immunoglobulin J539 [a mouse IgA2,K; anti-(1-',6)-/-D-galactan] was isolated and purified as described (5), and pepsin fragments were obtained by using a published procedure (6). Useful heavy atom derivatives were obtained by soaking J539 Fab crystals in stabilizing solutions with 0.25 mM K2Pt(CNS)6 for 2 weeks, 1 mM K2PtCl4 for 1 week, 0.3 mM K2HgI4 for 2 weeks, and 50 mM KI/1 mM chloramine-T (Eastman Kodak) for 1 week. The iodine-derivatized crystals were washed with fresh stabilizing solution after the soaking period. Unique sets of three-dimensional reflection data to 4.5-A spacings were collected from native and derivative crystals with a Picker FACS-1 diffractometer operated in the T-scan mode and using Cu Ka radiation. The various data sets were placed on the same relative scale, and pseudo temperature factors were applied to correct for differences in intensity fall-off. The K2Pt(CNS)6 derivative was analyzed first, and difference Patterson maps, readily interpretable, revealed one site of substitution (Fig. 1). After preliminary refinement with projection data only, the coordinates and occupancy of the K2Pt(CNS)6 site were used to determine the signs of the centric native protein reflections. These signs were used to examine the K2PtCI4 and K2HgI4 derivatives by difference Fourier analysis. The variables for these three heavy atom derivatives were then subjected to alternating cycles of least squares refinement and phase determination with the three-dimensional data. The resulting phasesThe publication costs of this article were defrayed in part by page charge paym...

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Section: Methodssupporting

confidence: 57%

Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5-A resolution.

Navia¹,

Segal²,

Padlan³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Possible factors include the overall charge, differences in amino-acid composition etc. The complexity of identifying the residues responsible is illustrated by the results on sickle-cell haemoglobin (Wishner, Ward, Lattman & Love, 1975) where a single substitution greatly affects the solubility with gruesome side affects.…”

Section: Physicochemical Properties Of Sbmepmentioning

confidence: 99%

Complex between the subtilisin from a mesophilic bacterium and the Leech inhibitor eglin-C

Dauter

Betzel²,

Genov³

et al. 1991

Acta Crystallogr Sect B

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“…1). This interaction allows deoxy-HbS to polymerize in an entropy-driven process (3)(4)(5). The polymerization of deoxyHbS leads to erythrocyte deformation from a biconcave morphology into the sickle shapes for which SCD is named.…”

mentioning

confidence: 99%

A Recombinant Human Hemoglobin with Anti-sickling Properties Greater than Fetal Hemoglobin

Levasseur

Ryan

Reilly

et al. 2004

Journal of Biological Chemistry

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A new recombinant, human anti-sickling ␤-globin polypeptide designated ␤ AS3 (␤Gly 16 3 Asp/␤Glu 22 3 Ala/␤Thr 87 3 Gln) was designed to increase affinity for ␣-globin. The amino acid substitutions at ␤22 and ␤87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxyHbS polymerization. The ␤16 substitution confers the recombinant ␤-globin subunit (␤ AS3 ) with a competitive advantage over ␤ S for interaction with the ␣-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (␣ 2 ␤ AS3 2 ) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that ␤ AS3 has a distinct advantage over ␤ S for dimerization with ␣-globin. When equal amounts of ␤ S -and ␤ AS3 -globin monomers compete for limiting ␣-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the ␤ AS3 polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of ␤ AS3 -globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit ␣-thalassemia. Therefore, constructs expressing the ␤ AS3 -globin gene may be suitable for future clinical trials for sickle cell disease.Sickle cell disease (SCD) 1 results from an A to T transversion at the sixth codon of the human ␤-globin gene on chromosome 11 (1, 2). The mutation of a single DNA base leads to the substitution of a valine for a glutamic acid in the ␤-globin polypeptide of sickle hemoglobin (HbS). The positioning of a hydrophobic residue at this position permits an interaction with a hydrophobic pocket on another hemoglobin tetramer (see Fig. 1). This interaction allows deoxy-HbS to polymerize in an entropy-driven process (3-5). The polymerization of deoxyHbS leads to erythrocyte deformation from a biconcave morphology into the sickle shapes for which SCD is named.Polymerization of deoxy-HbS is effectively inhibited by fetal hemoglobin (HbF), and individuals who are homozygous for the sickle mutation but also express high levels of HbF are typically asymptomatic (6 -8). The level of HbF necessary to significantly reduce the symptoms of SCD ranges from about 20 to 25%; however, data showing an enhanced red cell survival with as little as 9% HbF have been reported (9). The efficacy of HbF in inhibiting HbS polymerization suggests that transduction of fetal globin genes into hematopoietic stem cells might be an effective strategy for sickle cell disease gene therapy. However, high levels of ␥-globin gene expression are difficult to achieve in adult erythroid cells even...

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Crystal structure of sickle-cell deoxyhemoglobin at 5 Å resolution

Cited by 336 publications

References 20 publications

Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5-A resolution.

Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5-A resolution.

Complex between the subtilisin from a mesophilic bacterium and the Leech inhibitor eglin-C

A Recombinant Human Hemoglobin with Anti-sickling Properties Greater than Fetal Hemoglobin

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