Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

Abstract

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.

Solution structure of the variant‐3 neurotoxin from Centruroides sculpturatus Ewing

“…The methodology of protein structure refinement is based on the original work of Clore and Gronenborn and their coworkers (Nilges et al, 1988a,b,c;Driscoll et al, 1989) but with minor modifications as follows. Metric distance geometry algorithm (Crippen and Havel, 1978;Kuntz et al, 1979;Havel et al, 1983) was used to convert distances into Cartesian coordinates.…”

“…The potential energy (target function) consisted of terms for covalent, repulsion, NOE and torsion angle restraints. The forms of the target functions for the NOE and torsion angle constraints are chosen to be flat bottom potentials with harmonic potentials for the sides, and are identical to the ones used by Driscoll et al (1989). The modeling protocol used by us consists of four separate stages, viz.…”

“…(I) initial regularization, (11) regularization with refinement, (111) a long annealing step, and finally (IV) release of experimental constraints. The first stage (I) of this protocol consists of the four separate phases (Driscoll et al, 1989) and involves 200 cycles of Powell minimization (with kNoE = 0, k,, = 0, and kvdw = 4.19X10' kJ mol-' nmP4), a high-temperature (1000 K) molecular dynamics for 3.75 ps (where the force constants were varied in small steps, k,,, = 4.19X102 -2O9.5X1O2 kJ mol-' nm-', k,,, = 20.95 -838 kJ mol-' rad-'; kvdW = 419 -13.4X104kJ mo1-I nm-4), an annealing step consisting of 3-ps dynamics during which the temperature was decreased from 1000 K to 300 K in 25-K steps, and a 200-cycle Powell minimization with constraints. The second stage also consists of the same four phases, except that the force constants are modified in the following manner: the kNoE value was retained at 2.095XlO' kT mol-' nm-' during all four phases; the k,, value was kept at 20.95 kJ mol-' rad during the first two phases and at 838 kJ mol-' rad-' during the last two phases; the kvdw was 2.095X lo4 kJ mol-' nm-4 during phase 1, varied in increments from O.0126X1O4 to 16.76X104 kJ mol-I nmin phases 2 and 3, and maintained at 16.76X104 kJ mol-' nm-4 in phase 4.…”

Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two‐dimensional NMR spectroscopy

“…The refinement was carried out using only upper bounds as distance constraints while retaining all dihedral angle constraints. The annealing protocol was identical to the method described for refinement of BDS-I (Driscoll et al, 1989), except slow cooling was carried out in phase 3. Briefly, the protocol consists of 200 steps of Powell minimization during phase 1 followed by phase 2 , which consists of 3.75 ps of molecular dynamics at l , OOO K. During this dynamics phase, the NOE and dihedral angle force constants were increased from 0.5 to 50 kcal mol" A ' and 2.5 to 200 kcal mol" rad-2, respectively, by doubling the values every 75 ps.…”

“…The protocol was completed in phase 4 by performing 200 cycles of Powell minimization. All other details are identical to the BDS-1 protocol (Driscoll et al, 1989) except that the force constant for maintaining bond lengths was 1,000 kcal mol" A" instead of 600 kcal mol" Ap2.…”