Crystal structure at 1.95 A resolution of the breast tumour-specific antibody SM3 complexed with its peptide epitope reveals novel hypervariable loop recognition.

The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering.

Crystallization of an intact GST-estrogen receptor hormone binding domain fusion protein.

Crystals of an intact GST-estrogen receptor hormone binding domain fusion protein have been grown from solutions of MPD. The crystals grew as clusters of thin plates and needles of maximum dimensions 100 x 20 x 1 micrometer but were unsuitable for X-ray diffraction analysis. However, examination by electron microscopy shows an ordered lattice in which the protein molecules are clearly visible. Image analysis of electron micrographs of the protein crystals revealed electron stain-excluding density which showed a two-domain trimeric structure in projection, with each molecule of dimensions 12.0 x 5.0 nm diameter. The use of GST-fusion proteins in crystallisation are discussed.

Crystallization of the Fab fragment of the tumour-specific antibody PR1A3.

PR1A3 antibody binds specifically to the tumour-associated cell-surface antigen, carcinoembryonic antigen. Crystals of the Fab fragment of the PR1A3 antibody were obtained by vapour diffusion against mother liquor containing Tris-HC1 buffer, pH 8.6, magnesium chloride and polyethylene glycol 4000 as precipitating agent. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 42.2, b = 216.7, c = 45.9 A and beta = 95.6 degrees. Two Fab fragments are proesent in the asymmetric unit. Diffracted intensities up to 2.9 A resolution have been measured from frozen crystals.

Crystallization of an antitumour antibody SM3 complexed with a peptide epitope.

SM3 antibody binds to a tumour-associated epitope on polymorphic epithelial mucin (PEM). Crystals of the Fab fragment of SM3 in complex with a peptide antigen were obtained by vapour diffusion against mother liquor containing acetate buffer, pH 6.5, cadmium chloride and polyethylene glycol (PEG) 4000 as precipitating agent. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 42.2, b = 83.9, c = 64.5 A and beta = 93.4 degrees. One Fab-antigen complex is present in the asymmetric unit. Diffracted intensities up to 1.95 A resolution have been measured from a frozen crystal using synchrotron radiation.

In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.

Novel topology of a zinc-binding domain from a protein involved in regulating early Xenopus development.

Xenopus nuclear factor XNF7, a maternally expressed protein, functions in patterning of the embryo. XNF7 contains a number of defined protein domains implicated in the regulation of some developmental processes. Among these is a tripartite motif comprising a zinc-binding RING finger and B-box domain next to a predicted alpha-helical coiled-coil domain. Interestingly, this motif is found in a variety of protein including several proto-oncoproteins. Here we describe the solution structure of the XNF7 B-box zinc-binding domain determined at physiological pH by 1H NMR methods. The B-box structure represents the first three-dimensional structure of this new motif and comprises a monomer have two beta-strands, two helical turns and three extended loop regions packed in a novel topology. The r.m.s. deviation for the best 18 structures is 1.15 A for backbone atoms and 1.94 A for all atoms. Structure calculations and biochemical data shows one zinc atom ligated in a Cys2-His2 tetrahedral arrangement. We have used mutant peptides to determine the metal ligation scheme which surprisingly shows that not all of the seven conserved cysteines/histidines in the B-box motif are involved in metal ligation. The B-box structure is not similar in tertiary fold to any other known zinc-binding motif.

Antimicrobial prophylaxis for ophthalmic surgery.

Preoperative, intraoperative, and postoperative antibiotic agents have been used by ophthalmic surgeons routinely as prophylaxis for postoperative endophthalmitis. The rationale for such prophylaxis and the evidence which supports its efficacy are well founded. The optimal choice of antibiotic agent--from the standpoint of efficacy, route of delivery, adverse reactions, and cost--is far less established. This review considers these issues, as well as the role of topical disinfectants, including povidone-iodine, in preoperative prophylaxis.

Lower anxiety in today's high 'sue-ciety'.

Physician malpractice claims are part of the reason for the increased level of health care costs in the United States, according to authors Susan A. Chesteen, Ph.D., Fikry S. Gahin, Ph.D., and Joan M. Lally, Ph.D., of the University of Utah David Eccles School of Business. They present the results of a survey conducted on the residents of Utah on malpractice and discuss the implications for health care providers.

Characterisation of a novel cysteine/histidine-rich metal binding domain from Xenopus nuclear factor XNF7.

A 42 amino acid synthetic peptide corresponding to a newly defined cysteine/histidine-rich protein motif called B-box, from the Xenopus protein XNF7 has been characterised. The metal-binding stoichiometry and dissociation constant for zinc were determined by competition with the chromophoric chelator Br2BAPTA, demonstrating that one zinc atom binds per molecule of peptide despite the presence of seven putative metal ligands, and represents the first application of this method to measuring zinc stoichiometry of proteins and/or peptides. Cobalt binding studies indicate that the motif binds zinc more tightly than cobalt, that cysteines are used as ligands and that the cation is co-ordinated tetrahedrally. Circular dichroism and NMR studies both indicate that the B-box peptide is structured only in the presence of zinc, copper and to a lesser extent cobalt.

Lasers in cataract surgery.

Several laser technologies including 1053-nm picosecond, excimer, erbium:yttrium-aluminum-garnet, and neodymium:yttrium-aluminum-garnet (Nd:YAG) have been studied for use in the removal of cataractous lens tissue. Nd:YAG laser phacolysis involves laser pulses striking a titanium target that are incorporated into an irrigation-aspiration probe. With further refinement, Nd:YAG laser phacolysis may provide an alternative means of lens removal through a small incision, however, further study is required.