spread interest in this area. Notable developments include the The enediyne chromoproteins are a class of potent antitumour discovery of non-natural ligands for the apoproteins and the ob- antibiotics comprising a 1:1 complex of a protein and a nonco- servation that multiple binding modes are available for these li- valently bound chromophore. The protein is required to protect gands in the binding site. Mutation studies on the apoproteins and transport the highly labile chromophore, which acts as the have revealed much about their stability and variability, and the cytotoxic component by reacting with DNA leading to strand application of an in vitro evolution method has conferred new cleavage. A derivative of the best-studied member of this class, binding specificity for unrelated ligands. These investigations neocarzinostatin (NCS), is currently in use as a chemotherapeutic hold great promise for the application of the apoproteins for in Japan. The application of the chromoproteins as therapeutics drug-delivery, transport and stabilisation systems. along with their unique mode of action has prompted wide-

The natural complex Neocarzinostatin comprises a labile chromophore noncovalently bound to an 11.2 kDa protein. We present the first high-resolution structure of a novel complex derived from the recombinant apoprotein bound to a non-natural synthetic chromophore. Fluorescence and nuclear magnetic resonance spectroscopy were used to probe the strength and location of binding. Binding occurred in a location similar to that observed for the chromophore in the natural Neocarzinostatin complex, but with a distinct orientation. These results provide structural evidence that the apoprotein can readily accommodate small druglike entities, other than the natural chromophore within its binding cleft. The clinical use of the natural complex described by others, together with the results reported here, suggests potential applications for small molecule binding by apo-Neocarzinostatin

In this paper is described an analysis of the effects of protein flexibility on the observed CIS values and the impact on the accuracy of 3D structures determined using a 1H NMR CIS approach. The effects of protein conformational mobility have been investigated by using a set of different protein structures as starting points for the calculation: the unbound X-ray crystal structure, the unbound NMR solution structure, and the bound NMR solution structure of the protein. The results indicated that loop movement does have a significant impact on the quality of the structure generated by the CIS structure determination methodology. The implementation of methods to treat loop flexibility within our protocol, however, did not improve the results for calculations based on the unbound protein frame.

The new program DYANA (DYnamics Algorithm for Nmr Applications) for ef®cient calculation of three-dimensional protein and nucleic acid structures from distance constraints and torsion angle constraints col- lected by nuclear magnetic resonance (NMR) experiments performs simu- lated annealing by molecular dynamics in torsion angle space and uses a fast recursive algorithm to integrate the equations of motions. Torsion angle dynamics can be more ef®cient than molecular dynamics in Carte- sian coordinate space because of the reduced number of degrees of free- dom and the concomitant absence of high-frequency bond and angle vibrations, which allows for the use of longer time-steps and/or higher temperatures in the structure calculation. It also represents a signi®cant advance over the variable target function method in torsion angle space with the REDAC strategy used by the predecessor program DIANA. DYANA computation times per accepted conformer in the ``bundle'' used to rep- resent the NMR structure compare favorably with those of other pre- sently available structure calculation algorithms, and are of the order of 160 seconds for a protein of 165 amino acid residues when using a DEC Alpha 8400 5/300 computer. Test calculations starting from conformers with random torsion angle values further showed that DYANA is capable of ef®cient calculation of high-quality protein structures with up to 400 amino acid residues, and of nucleic acid structures.

The solution structure of diadenosine 5H , 5HHH -P1, P4-tetraphosphate hydro- Department of Biochemistry and Molecular Biology lase from Lupinus angustifolius L., an enzyme of the Nudix family, has University of Melbourne been determined by heteronuclear NMR, using a torsion angle Parkville, Victoria dynamics/simulated annealing protocol based on approximately 12 inter- residue NOEs per residue. The structure represents the ®rst Ap4A hydro- 3010, Australia lase to be determined, and sequence homology suggests that other 2 Department of Chemistry members will have the same fold. The family of structures shows a well- University of Shef®eld de®ned fold comprised of a central four-stranded mixed b-sheet, a two- Shef®eld, S3 7HF, England stranded antiparallel b-sheet and three helices (aI, aIII, aIV). The root- mean-squared deviation for the backbone (CH , O, N, Ca) of the rigid parts Ê (residues 9 to 75, 97 to 115, 125 to 160) of the protein is 0.32 A. Several regions, however, show lower de®nition, particularly an isolated helix (aII) that connects two strands of the central sheet. This poor de®nition is mainly due to a lack of long-range NOEs between aII and other parts of the protein. Mapping conserved residues outside of the Nudix signature and those sensitive to an Ap4A analogue suggests that the adenosine- ribose moiety of the substrate binds into a large cleft above the four- stranded b-sheet. Four conserved glutamate residues (Glu55, Glu58, Glu59 and Glu125) form a cluster that most likely ligates an essential magnesium ion, however, Gly41 also an expected magnesium ligand, is distant from this cluster.

The proteins of the pancreatic ribonuclease A (RNase A) family catalyze the cleavage of the RNA polymer chain. The development of RNase inhibitors is of significant interest, as some of these compounds may have a therapeutic effect in pathological conditions associated with these proteins. The most potent low molecular weight inhibitor of RNase reported to date is the compound 5'-phospho-2'-deoxyuridine-3-pyrophosphate (P->5)-adenosine-3-phosphate (pdUppA-3'-p). The 3',5'-pyrophosphate group of this compound increases its affinity and introduces structural features which seem to be unique in pyrophosphate-containing ligands bound to RNase A, such as the adoption of a syn conformation by the adenosine base at RNase subsite B2 and the placement of the 5'-ß-phosphate of the adenylate (instead of the {alpha}-phosphate) at subsite P1 where the phosphodiester bond cleavage occurs. In this work, we study by multi-ns molecular dynamics simulations the structural properties of RNase A complexes with the ligand pdUppA-3'-p and the related weaker inhibitor dUppA, which lacks the 3' and 5' terminal phosphate groups of pdUppA-3'-p. The simulations show that the adenylate 5'-ß-phosphate binding position and the adenosine syn orientation constitute robust structural features in both complexes, stabilized by persistent interactions with specific active-site residues of subsites P1 and B2. The simulation structures are used in conjunction with a continuum-electrostatics (Poisson-Boltzmann) model, to evaluate the relative binding affinity of the two complexes. The computed relative affinity of pdUppA-3'-p varies between –7.9 kcal/mol and –2.8 kcal/mol for a range of protein/ligand dielectric constants ({epsilon}p) 2–20, in good agreement with the experimental value (–3.6 kcal/mol); the agreement becomes exact with {epsilon}p = 8. The success of the continuum-electrostatics model suggests that the differences in affinity of the two ligands originate mainly from electrostatic interactions. A residue decomposition of the electrostatic free energies shows that the terminal phosphate groups of pdUppA-3'-p make increased interactions with residues Lys7 and Lys66 of the more remote sites P2 and P0, and His119 of site P1.

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