Lessons Learned: Early Termination of a Randomized Trial of Calcineurin Inhibitor and Corticosteroid Avoidance Using Belatacept.

The intent of this National Institutes of Health-sponsored study was to compare a belatacept-based immunosuppressive regimen with a maintenance regimen of tacrolimus and mycophenolate. Nineteen primary, Epstein-Barr virus-immune renal transplant recipients with a negative cross-match were randomized to one of three groups. All patient groups received perioperative steroids and maintenance mycophenolate mofetil. Patients in groups 1 and 2 were induced with alemtuzumab and maintained on tacrolimus or belatacept, respectively. Patients in group 3 were induced with basiliximab, received 3 mo of tacrolimus, and maintained on belatacept. There was one death with a functioning allograft due to endocarditis (group 1). There were three graft losses due to vascular thrombosis (all group 2) and one graft loss due to glomerular disease (group 1). Biopsy-proven acute cellular rejection was more frequent in the belatacept-treated groups, with 10 treated episodes in seven participants compared with one episode in group 1; however, estimated GFR was similar between groups at week 52. There were no episodes of posttransplant lymphoproliferative disorder or opportunistic infections in any group. Protocol enrollment was halted prematurely because of a high rate of serious adverse events. Such negative outcomes pose challenges to clinical investigators, who ultimately must weigh the risks and benefits in randomized trials.

Exploring the limits of precision and accuracy of protein structures determined by nuclear magnetic resonance spectroscopy.

The effects of the number, precision and accuracy of interproton distance restraints, of direct refinement against nuclear Overhauser enhancement (NOE) intensities and of the description of the non-bonded contacts on the precision and accuracy of a nuclear magnetic resonance (NMR) protein structure determination have been investigated. The model system employed is the 56 residue immunoglobulin G binding domain of streptococcal protein G. This choice was based on the availability of a very high resolution NMR structure (atomic root-mean-square distribution of the ensemble of 60 calculated structures about the mean co-ordinate positions of 0.25 A for the backbone atoms, 0.65 A for all atoms and 0.39 A for all atoms excluding disordered surface side-chains). The experimental NMR data set for this structure determination comprised a total of 1058 experimental restraints of which 854 were approximate interproton distance restraints corresponding to all the structurally useful NOEs observable for this protein. The calculations presented in this paper reveal the following. (1) The number of interproton distance restraints constitutes the single most important determinant of both precision and accuracy. The ensemble precision and accuracy improves significantly as the number of interproton distance restraints is increased to an average of approximately 15 per residue, of which approximately 60% involve unique proton pairs; subsequent additions of interproton distance restraints, however, lead to less dramatic improvements as information redundancy sets in. (2) The ratio of ensemble precision to ensemble accuracy (which ranges from 0.5 to 0.7 for the backbone atoms) is approximately independent both of the number, precision and accuracy of the interproton distance restraints, and of whether the structures are refined against interproton distance restraints or directly against NOE intensities. (3) In an ensemble of structures generated from a large number of loose approximate interproton distance restraints (an average of approximately 15 restraints per residue with approximately 60% involving unique proton pairs), the interproton distance vectors corresponding to the restraints are very well defined with approximately 80% of vectors between unique proton pairs having a standard deviation of < or = 0.1 A. (4) The accuracy of the mean co-ordinates of an ensemble of structures is significantly higher than the average accuracy of the individual structures comprising the ensemble. For an average ensemble precision of > or = 0.6 A, the dependence of the accuracy of the mean co-ordinates on ensemble precision is approximately linear.(ABSTRACT TRUNCATED AT 400 WORDS)

Three-dimensional solution structure of the E3-binding domain of the dihydrolipoamide succinyltransferase core from the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli.

The three-dimensional solution structure of a 51-residue synthetic peptide comprising the dihydrolipoamide dehydrogenase (E3)-binding domain of the dihydrolipoamide succinyltransferase (E2) core of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli has been determined by nuclear magnetic resonance spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure is based on 630 approximate interproton distance and 101 torsion angle (phi, psi, chi 1) restraints. A total of 56 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions for residues 12-48 of the synthetic peptide is 1.24 A for the backbone atoms, 1.68 A for all atoms, and 1.33 A for all atoms excluding the six side chains which are disordered at chi 1 and the seven which are disordered at chi 2; when the irregular partially disordered loop from residues 31 to 39 is excluded, the rms distribution drops to 0.77 A for the backbone atoms, 1.55 A for all atoms, and 0.89 A for ordered side chains. Although proton resonance assignments for the N-terminal 11 residues and the C-terminal 3 residues were obtained, these two segments of the polypeptide are disordered in solution as evidenced by the absence of nonsequential nuclear Overhauser effects. The solution structure of the E3-binding domain consists of two parallel helices (residues 14-23 and 40-48), a short extended strand (24-26), a five-residue helical-like turn, and an irregular (and more disordered) loop (residues 31-39). This report presents the first structure of an E3-binding domain from a 2-oxo acid dehydrogenase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution solution structure of the double Cys2His2 zinc finger from the human enhancer binding protein MBP-1.

The high-resolution three-dimensional structure of a synthetic 57-residue peptide comprising the double zinc finger of the human enhancer binding protein MBP-1 has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1280 experimental restraints. A total of 30 simulated annealing structures were calculated. The backbone atomic root-mean-square distributions about the mean coordinate positions are 0.32 and 0.33 A for the N- and C-terminal fingers, respectively, and the corresponding values for all atoms, excluding disordered surface side chains, are 0.36 and 0.40 A. Each finger comprises an irregular antiparallel sheet and a helix, with the zinc tetrahedrally coordinated to two cysteines and two histidines. The overall structure is nonglobular in nature, and the angle between the long axes of the helices is 47 +/- 5 degrees. The long axis of the antiparallel sheet in the N-terminal finger is approximately parallel to that of the helix in the C-terminal finger. Comparison of this structure with the X-ray structure of the Zif-268 triple finger complexed with DNA indicates that the relative orientation of the individual zinc fingers is clearly distinct in the two cases. This difference can be attributed to the presence of a long Lys side chain in the C-terminal finger of MBP-1 at position 40, instead of a short Ala or Ser side chain at the equivalent position in Zif-268. This finding suggests that different contacts may be involved in the binding of the zinc fingers of MBP-1 and Zif-268 to DNA, consistent with the findings from methylation interference experiments that the two fingers of MBP-1 contact 10 base pairs, while the three fingers of Zif-268 contact only 9 base pairs.