Chiral protein scissors: High enantiomeric selectivity for binding and its effect on protein photocleavage efficiency and specificity

Chiral recognition of protein-binding sites by a simple organic molecule with selectivities >100 is reported here. The l-isomer of 4(1-pyrene)-3-butyroyl-phenylalanine amide (Py-l-Phe) binds to BSA with an affinity constant (K(b)) of 3 × 10(7) M(−1), whereas the corresponding d-isomer (Py-d-Phe) binds 100 times weaker. The enantiomers showed contrasting spectral changes when bound to BSA. Whereas hypochromism was observed with the l-isomer, hyperchromism was observed for the d-isomer, and, whereas the fluorescence of the l-isomer was quenched, the fluorescence of the d-isomer was enhanced. The induced CD spectra of the enantiomers bound to BSA bear a near mirror-image relationship. In contrast, the enantiomers show only moderate binding selectivity with lysozyme. The differences in the enantioselectivities with the two proteins indicate that the binding site of 4(1-pyrene)-3-butyroyl-phenylalanine amide (Py-Phe) in BSA is crowded, whereas that of lysozyme is more accommodative of either isomer. The enantioselective binding of Py-Phe isomers is further examined in protein photocleavage studies. Py-d-Phe cleaves BSA and lysozyme at a single site in a manner similar to Py-l-Phe, but the cleavage yields are lower for the d-isomer. Sequencing of the resulting fragments indicated that the photocleavage sites of Py-d-Phe on BSA and lysozyme are identical to those of Py-l-Phe. Flash photolysis studies indicated only minor differences between the two enantiomers. The large binding selectivities, therefore, do not influence cleavage specificity or cleavage site location. The strong role of the single asymmetric center of Py-Phe in recognition and its minor role in photocleavage chemistry are demonstrated

The chaperones MPP11 and Hsp70L1 form the mammalian ribosome-associated complex

Soluble Hsp70 homologs cotranslationally interact with nascent polypeptides in all kingdoms of life. In addition, fungi possess a specialized Hsp70 system attached to ribosomes, which in Saccharomyces cerevisiae consists of the Hsp70 homologs Ssb1/2p, Ssz1p, and the Hsp40 homolog zuotin. Ssz1p and zuotin are assembled into a unique heterodimeric complex termed ribosome-associated complex. So far, no such specialized chaperones have been identified on ribosomes of higher eukaryotes. However, a family of proteins characterized by an N-terminal zuotin-homology domain fused to a C-terminal two-repeat Myb domain is present in animals and plants. Members of this family, like human MPP11 and mouse MIDA1, have been implicated in the regulation of cell growth. Specific targets of MPP11/MIDA1, however, have remained elusive. Here, we report that MPP11 is localized to the cytosol and associates with ribosomes. Purification of MPP11 revealed that it forms a stable complex with Hsp70L1, a distantly related homolog of Ssz1p. Complementation experiments indicate that mammalian ribosome-associated complex is functional in yeast. We conclude that despite a low degree of homology on the amino acid level cooperation of ribosome-associated chaperones with the translational apparatus is well conserved in eukaryotic cells

Dimeric organization of the yeast oligosaccharyl transferase complex

The enzyme complex oligosaccharyl transferase (OT) catalyzes N-glycosylation in the lumen of the endoplasmic reticulum. The yeast OT complex is composed of nine subunits, all of which are transmembrane proteins. Several lines of evidence, including our previous split-ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one-step immunoaffinity technique. The digitonin-solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of ≈500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25-Å resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation

Dissection of a viral autoprotease elucidates a function of a cellular chaperone in proteolysis

Replication of positive-strand RNA viruses involves translation of polyproteins which are proteolytically processed into functional peptides. These maturation steps often involve virus-encoded autoproteases specialized in generating their own N or C termini. Nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus represents such an enzyme. Bovine viral diarrhea virus NS2 creates in cis its own C terminus and thereby releases an essential viral replication factor. As a unique feature, this enzyme requires for proteolytic activity stoichiometric amounts of a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) or its fragment Jiv90. To obtain insight into the structural organization of the NS2 autoprotease, the basis for its restriction to cis cleavage, as well as its activation by Jiv, we dissected NS2 into functional domains. Interestingly, an N-terminal NS2 fragment covering the active center of the protease, cleaved in trans an artificial substrate composed of a C-terminal NS2 fragment and two downstream amino acids. In the authentic NS2, the 4 C-terminal amino acids interfered with binding and cleavage of substrates offered in trans. These findings strongly suggest an intramolecular product inhibition for the NS2 autoprotease. Remarkably, the chaperone fragment Jiv90 independently interacted with protease and substrate domain and turned out to be essential for the formation of a protease/substrate complex that is required for cleavage. Thus, the function of the cell-derived protease cofactor Jiv in proteolysis is regulation of protease/substrate interaction, which ultimately results in positioning of active site and substrate peptide into a cleavage-competent conformation

A βα-barrel built by the combination of fragments from different folds

Combinatorial assembly of protein domains plays an important role in the evolution of proteins. There is also evidence that protein domains have come together from stable subdomains. This concept of modular assembly could be used to construct new well folded proteins from stable protein fragments. Here, we report the construction of a chimeric protein from parts of a (βα)8-barrel enzyme from histidine biosynthesis pathway (HisF) and a protein of the (βα)5-flavodoxin-like fold (CheY) from Thermotoga maritima that share a high structural similarity. We expected this construct to fold into a full (βα)8-barrel. Our results show that the chimeric protein is a stable monomer that unfolds with high cooperativity. Its three-dimensional structure, which was solved to 3.1 Å resolution by x-ray crystallography, confirms a barrel-like fold in which the overall structures of the parent proteins are highly conserved. The structure further reveals a ninth strand in the barrel, which is formed by residues from the HisF C terminus and an attached tag. This strand invades between β-strand 1 and 2 of the CheY part closing a gap in the structure that might be due to a suboptimal fit between the fragments. Thus, by a combination of parts from two different folds and a small arbitrary fragment, we created a well folded and stable protein

A redox-active FKBP-type immunophilin functions in accumulation of the photosystem II supercomplex in Arabidopsis thaliana

Photosystem II (PSII) catalyzes the first of two photosynthetic reactions that convert sunlight into chemical energy. Native PSII is a supercomplex consisting of core and light-harvesting chlorophyll proteins. Although the structure of PSII has been resolved by x-ray crystallography, the mechanism underlying its assembly is poorly understood. Here, we report that an immunophilin of the chloroplast thylakoid lumen is required for accumulation of the PSII supercomplex in Arabidopsis thaliana. The immunophilin, FKBP20-2, belongs to the FK-506 binding protein (FKBP) subfamily that functions as peptidyl-prolyl isomerases (PPIases) in protein folding. FKBP20-2 has a unique pair of cysteines at the C terminus and was found to be reduced by thioredoxin (Trx) (itself reduced by NADPH by means of NADP-Trx reductase). The FKBP20-2 protein, which contains only two of the five amino acids required for catalysis, showed a low level of PPIase activity that was unaffected on reduction by Trx. Genetic disruption of the FKBP20-2 gene resulted in reduced plant growth, consistent with the observed lower rate of PSII activity determined by fluorescence (using leaves) and oxygen evolution (using isolated chloroplasts). Analysis of isolated thylakoid membranes with blue native gels and immunoblots showed that accumulation of the PSII supercomplex was compromised in mutant plants, whereas the levels of monomer and dimer building blocks were elevated compared with WT. The results provide evidence that FKBP20-2 participates specifically in the accumulation of the PSII supercomplex in the chloroplast thylakoid lumen by means of a mechanism that has yet to be determined

Mimicking enzyme evolution by generating new (βα)(8)-barrels from (βα)(4)-half-barrels

Gene duplication and fusion events that multiply and link functional protein domains are crucial mechanisms of enzyme evolution. The analysis of amino acid sequences and three-dimensional structures suggested that the (βα)(8)-barrel, which is the most frequent fold among enzymes, has evolved by the duplication, fusion, and mixing of (βα)(4)-half-barrel domains. Here, we mimicked this evolutionary strategy by generating in vitro (βα)(8)-barrels from (βα)(4)-half-barrels that were deduced from the enzymes imidazole glycerol phosphate synthase (HisF) and N′[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide isomerase (HisA). To this end, the gene for the C-terminal (βα)(4)-half-barrel (HisF-C) of HisF was duplicated and fused in tandem to yield HisF-CC, which is more stable than HisF-C. In the next step, by optimizing side-chain interactions within the center of the β-barrel of HisF-CC, the monomeric and compact (βα)(8)-barrel protein HisF-C*C was generated. Moreover, the genes for the N- and C-terminal (βα)(4)-half-barrels of HisF and HisA were fused crosswise to yield the chimeric proteins HisFA and HisAF. Whereas HisFA contains native secondary structure elements but adopts ill-defined association states, the (βα)(8)-barrel HisAF is a stable and compact monomer that reversibly unfolds with high cooperativity. The results obtained suggest a previously undescribed dimension for the diversification of enzymatic activities: new (βα)(8)-barrels with novel functions might have evolved by the exchange of (βα)(4)-half-barrel domains with distinct functional properties