....Protein Data Bank...

...InTroduction....

For simplicity, one can think of the PDB as a database of protein- and nucleic acid structures. However, it is important to keep in mind that the PDB entries are actually descriptions of structure determination experiments and their results. While such experiments usually aim at determining the structure of a protein or other macromolecule, the result of the experiment is actually just a model of the physical molecule.

The PDB was one of the first central repositories for biological data, preceding similar databases like Genbank for genomic sequences or Pubmed for biomedical literature. It was also one of the first examples where publication authors were required to submit experimental data to a central database before publication [5]. The PDB is now the single worldwide archive of structural data of biological macromolecules [3].

...HistoRY...

The PDB was initiated in 1971 by the Brookhaven National Laboratory in New York [2]. Some authors still refer to it as the Brookhaven Protein Data Bank even though it has not been associated with Brookhaven since 1999. Since this time the PDB has been maintained jointly by the Research Collaboratory for Structural Bioinformatics (RCSB), PDBe at the European Bioinformatics Institute (EBI), the PDBj in Japan and the BMRB (USA). These now form the Worldwide Protein Data Bank (wwPDB) organization, which was announced in 2003 [3]. The members of the wwPDB are maintaining and developing the PDB as the single and publicly available archive of macromolecular structural data.

...Ftsh PEptiDasE....

Authors: Han.S Experiment:X-Ray Diffraction with resolution of 2.00A Chain:A

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.

...THemolysin...

Authors:   English, A.C.,   Groom, C.R.,    Hubbard, R.E. Experiment:X-ray Diffraction With Resolution of 2.00A

     Chains:A

Thermolysin has a molecular weight of 34,600 Da. Its overall structure consists of two roughly spherical domains with a deep cleft running across the middle of the molecule separating the two domains. The secondary structure of each domain is quite different, the N-terminal domain consists of mostly beta pleated sheet, while the C-terminal domain is mostly alpha helical in structure. These two domains are connected by a central alpha helix, spanning amino acids 137-151.^[7]

In contrast to many proteins that undergo conformational changes upon heating and denaturation, thermolysin does not undergo any major conformational changes until at least 70 °C.^[8] The thermal stability of members of the TLP family is measured in terms of a T₅₀ temperature. At this temperature incubation for 30 minutes reduces the enzymes activity by half. Thermolysin has a T₅₀ value of 86.9 °C, making it the most thermo stable member of the TLP family.^[9] Studies on the contribution of calcium to thermolysin stability have shown that upon thermal inactivation a single calcium ion is released from the molecule.^[10] Preventing this calcium from originally binding to the molecule by mutation of its binding site, reduced thermolysin stability by 7 °C. However, while calcium binding makes a significant contribution to stabilising thermolysin, more crucial to stability is a small cluster of N-terminal domain amino acids located at the proteins surface.^[9] In particular a phenylalanine (F) at amino acid position 63 and a proline (P) at amino acid position 69 contribute significantly to thermolysin stability. Changing these amino acids to threonine (T) and alanine (A) respectively in a less stable thermolysin-like proteinase produced by Bacillus stearothermophillus (TLP-ste), results in individual reductions in stability of 7 °C (F63→T) and 6.3 °C (P69→A) and when combined a reduction in stability of 12.3 °C.^[9]

 ...LEuCYL AminOPePtiDase.....

 

Authors:

Kale, A.,   Dijkstra, B.W.,   Sonke, T.,   Thunnissen, A.M.W.H

Experimet:X-ray Diffraction with Resolution of 2.75A

Chain:A,B

 The zinc-dependent leucine aminopeptidase from Pseudomonas putida (ppLAP) is an important enzyme for the industrial production of enantiomerically pure amino acids. To provide a better understanding of its structure-function relationships, the enzyme was studied by X-ray crystallography. Crystal structures of native ppLAP at pH 9.5 and pH 5.2, and in complex with the inhibitor bestatin, show that the overall folding and hexameric organization of ppLAP are very similar to those of the closely related di-zinc leucine aminopeptidases (LAPs) from bovine lens and Escherichia coli. At pH 9.5, the active site contains two metal ions, one identified as Mn(2+) or Zn(2+) (site 1), and the other as Zn(2+) (site 2). By using a metal-dependent activity assay it was shown that site 1 in heterologously expressed ppLAP is occupied mainly by Mn(2+). Moreover, it was shown that Mn(2+) has a significant activation effect when bound to site 1 of ppLAP. At pH 5.2, the active site of ppLAP is highly disordered and the two metal ions are absent, most probably due to full protonation of one of the metal-interacting residues, Lys267, explaining why ppLAP is inactive at low pH. A structural comparison of the ppLAP-bestatin complex with inhibitor-bound complexes of bovine lens LAP, along with substrate modelling, gave clear and new insights into its substrate specificity and high level of enantioselectivity.