Mason-Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C(α) deviations are large and the active-site 'DTG' loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections, including AIDS.

Following the failure of a wide range of attempts to solve the crystal structure of M-PMV retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs.

The crystal structure of TeRbcX, a RuBisCO assembly chaperone from the cyanobacterium Thermosynechococcus elongatus, a thermophilic organism, has been determined at 1.7 Å resolution. TeRbcX has an unusual cysteine residue at position 103 that is not found in RbcX proteins from mesophilic organisms. Unlike wild-type TeRbcX, a mutant protein with Cys103 replaced by Ala (TeRbcX-C103A) could be readily crystallized. The structure revealed that the overall fold of the TeRbcX homodimer is similar to those of previously crystallized RbcX proteins. Normal-mode analysis suggested that TeRbcX might adopt an open or closed conformation through a hinge movement pivoted on a kink in two long α4 helices. This type of conformational transition is presumably connected to RbcL (the large RuBisCO subunit) binding during the chaperone function of the RuBisCO assembly.

The structure of the reduced form of cytochrome c(6) from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.2 A and refined to an R-factor of 0.107. This protein is unique among all known cytochromes c(6), owing to the presence of an unusual seven-residue insertion, KDGSKSL(44-50), which differs from the insertion found in the recently discovered plant cytochromes c(6A). Furthermore, the present protein is unusual because of its very high content (36%) of the smallest residues (glycine and alanine). The structure reveals that the overall fold of the protein is similar to that of other class I c-type cytochromes, despite the presence of the specific insertion. The insertion is located within the most variable region of the cytochrome c(6) sequence, i.e. between helices II and III. The first six residues [KDGSKS(44-49)] form a loop, whereas the last residue, Leu50, extends the N-terminal beginning of helix III. Several specific noncovalent interactions are found inside the insertion, as well as between the insertion and the rest of the protein. The crystal structure contains three copies of the cytochrome c(6) molecule per asymmetric unit, and is characterized by an unusually high packing density, with solvent occupying barely 17.58% of the crystal volume.

RbcX is a dimeric protein found in cyanobacteria that assists in the assembly of the oligomeric RuBisCO complex. RbcX from the thermophile Thermosynechococcus elongatus (TeRbcX) contains an unusual Cys103 residue in its sequence and when expressed recombinantly the protein aggregates and cannot be crystallized. Site-directed mutagenesis of Cys103 to either Arg or Ala produced non-aggregating proteins that could be readily crystallized in several crystal forms. Synchrotron-radiation X-ray diffraction data were collected to 1.96 A resolution and formed the basis of crystal structure analysis of TeRbcX.

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95 A structure of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD, which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part of the C-terminal extension (CTE) of the EcRDBD folds into an alpha-helix whose location in the minor groove does not match any of the locations previously observed for nuclear receptors. Mutational analyses suggest that the alpha-helix is a component of EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to be a general feature of all known EcRs.

Gankyrin is a 25-kDa hepatocellular carcinoma-associated protein that mediates protein-protein interactions in cell cycle control and protein degradation. It has been reported to form complexes with cyclin-dependent kinase 4, retinoblastoma protein, the S6b ATPase subunit of the 19 S regulator of the 26 S proteasome, and Mdm2, an E3 ubiquitin ligase involved in p53 degradation. It is the first protein described to bind both to the 26 S proteasome and to proteins in other complexes containing cyclin-dependent kinase(s) and p53 ubiquitylating activities, thus providing a mechanism for delivering cell cycle regulating machinery and ubiquitylated substrates to the proteasome for degradation. Gankyrin contains a 33-residue motif known as the ankyrin repeat that occurs five and a half to six times in the sequence. As a step toward understanding gankyrin interactions with its protein partners we have determined its three-dimensional crystal structure to 2.0-A resolution. It reveals that the entire 226-residue gankyrin polypeptide folds into seven ankyrin repeat elements. The ankyrin repeats, consisting of an antiparallel beta-hairpin followed by a perpendicularly oriented helix-loop-helix, pack side-by-side, creating an extended curved structure with a groove running across the long concave surface. Comparison with the structures of other ankyrin repeat proteins suggests that interactions with partner proteins are mediated by residues situated on this concave surface.

Gankyrin is an oncoprotein overexpressed in hepatocarcinoma cells that binds to the cell-cycle regulator CDK4 and the S6b ATPase subunit of the regulatory component of the proteasome. It belongs to the family of ankyrin-repeat proteins that appear to mediate protein-protein interactions in diverse biochemical processes. Gankyrin has been crystallized from polyethylene glycol solutions and diffraction data have been obtained from these crystals that extend to 2.1 A spacing.

Eubacteria and eukaryotic cellular organelles have membrane-bound ATP-dependent proteases, which degrade misassembled membrane protein complexes and play a vital role in membrane quality control. The bacterial protease FtsH also degrades an interesting subset of cytoplasmic regulatory proteins, including sigma(32), LpxC, and lambda CII. The crystal structure of the ATPase module of FtsH has been solved, revealing an alpha/beta nucleotide binding domain connected to a four-helix bundle, similar to the AAA modules of proteins involved in DNA replication and membrane fusion. A sulfate anion in the ATP binding pocket mimics the beta-phosphate group of an adenine nucleotide. A hexamer form of FtsH has been modeled, providing insights into possible modes of nucleotide binding and intersubunit catalysis.