Characterization and crystal structure of zinc azurin, a by‐product of heterologous expression in <i>Escherichia coli of Pseudomonas aeruginosa</i> copper azurin

Nar, Herbert; Huber, Robert; Messerschmidt, Albrecht; Filippou, Alexander C.; Barth, Manfred; Jaquinod, Michel; Kamp, Mart van de; Canters, Gerard W.

doi:10.1111/j.1432-1033.1992.tb16881.x

Cited by 147 publications

(169 citation statements)

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“…Among a large number of azurin-like crystal structures determined to date, a zinc ion-bound Paz (Zn-Paz) expressed in recombinant E. coli cells has been isolated and structurally characterized. 25 In the crystal structure of Zn-Paz (PDB code, 1E67), the distance between the zinc ion and Sd of Met141 is 3.31 A (Fig. 5B, right, blue), while the corresponding distance is 2.97 A in Cu-Paz (PDB code, 3AZU) (Fig.…”

Section: Overall Structurementioning

confidence: 99%

Structural studies on Laz, a promiscuous anticancer Neisserial protein

et al. 2015

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Azurin and Laz (lipidated azurin) are 2 bacterial proteins with anticancer, anti-viral and anti-parasitic activities. Azurin, isolated from the bacterium Pseudomonas aeruginosa, termed Paz, demonstrates anticancer activity against a range of cancers but not against brain tumors. In contrast, Laz is produced by members of Gonococci/Meningococci, including Neisseria meningitides which can cross the blood-brain barrier to infect brain meninges. It has been previously reported that Laz has an additional 39 amino acid moiety, called an H.8 epitope, in the N-terminal part of the azurin moiety that allows Laz to cross the entry barrier to brain tumors such as glioblastomas. Exactly, how the H.8 epitope helps the azurin moiety of Laz to cross the entry barriers to attack glioblastoma cells is unknown. In this paper, we describe the structural features of the H.8 moiety in Laz using X-ray crystallography and demonstrate that while the azurin moiety of Laz adopts a b-sandwich fold with 2 b-sheets arranged in the Greek key motif, the H.8 epitope was present as a disordered structure outside the Greek key motif. Structures of Paz and H.8 epitope-deficient Laz are well superimposed. The structural flexibility of the H.8 motif in Laz explains the extracellular location of Laz in Neisseria where it can bind the key components of brain tumor cells to disrupt their tight junctions and allow entry of Laz inside the tumors to exert cytotoxicity.

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Section: Overall Structurementioning

confidence: 99%

Structural studies on Laz, a promiscuous anticancer Neisserial protein

et al. 2015

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“…With heterologous expression the contamination may reach levels of 50% or more. The Zn protein is often difficult to remove and it requires special care to obtain preparations of Cu protein that are free from the contaminating Zn analog [49,50].…”

Section: Geneticsmentioning

confidence: 99%

Engineering type 1 copper sites in proteins

1993

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The use of site-directed mutagenesis methods has revolutionalized the study of the so-called type 1 and type 2 copper sites in proteins. In particular our understanding of the relation between the structure, and the mechanistic and spectroscopic features of these sites is benefitting from the application of these techniques. Recent progress in the field is reviewed with emphasis on the study of type 1 sites. Topics covered comprise the characteristics of the natural type 1 and type 2 sites, the genetics of blue copper proteins, the modification of Cu sites, the spectroscopy of natural and engineered type 1 and type 2 sites, the effect of mutations on midpoint potentials and the mechanism of electron transfer as carried out by the blue copper proteins.

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“…As a result, Cu 2ϩ metallated azurin does not fold reversibly in the laboratory (16); thus, a thorough investigation of how the metal center influences the protein's stability and folding dynamics is very difficult. Fortunately, Zn 2ϩ can be exchanged for copper without significant change to the rigid structure of azurin (14,17). Because zinc is essentially redox inactive, a more detailed assessment of the metal's role in the folding landscape can be performed for this system.…”

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confidence: 99%

Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin

Zong

Wilson

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Understanding how the folding of proteins establishes their functional characteristics at the molecular level challenges both theorists and experimentalists. The simplest test beds for confronting this issue are provided by electron transfer proteins. The environment provided by the folded protein to the cofactor tunes the metal's electron transport capabilities as envisioned in the entatic hypothesis. To see how the entatic state is achieved one must study how the folding landscape affects and in turn is affected by the metal. Here, we develop a coarse-grained functional to explicitly model how the coordination of the metal (which results in a so-called entatic or rack-induced state) modifies the folding of the metallated Pseudomonas aeruginosa azurin. Our free-energy functional-based approach directly yields the proper nonlinear extrathermodynamic free energy relationships for the kinetics of folding the wild type and several point-mutated variants of the metallated protein. The results agree quite well with corresponding laboratory experiments. Moreover, our modified free-energy functional provides a sufficient level of detail to explicitly model how the geometric entatic state of the metal modifies the dynamic folding nucleus of azurin.curved chevron ͉ cupredoxin ͉ metalloproteins T he entatic state occurs in proteins when a group, metal or nonmetal, is forced into an unusual, energetically strained geometric or electronic state (rack-induced state) (1-4). Through the polypeptide's folding-induced rigidity, the protein fails to provide the expected geometry of ligating groups that would occur with freely mobile ligands in solution, thereby tuning the ligand's redox characteristics. In metalloproteins, the metal ions are typically bound to the protein through one or more lone pair donors, endogenous biological ligands (e.g., the imidazole moiety of histidine, the carbonyl oxygen of the main chain or the side chain of an asparagine residue). In several cases the ligands are arranged such that an optimal geometry is precluded (1-4). The resulting entatic state in a given metalloprotein is determined by the entire rigid protein scaffold in concert with the hydrogen-bonding network proximal to the coordination sphere (5, 6). The particular geometry of the rack-induced state influences the electronic structure of the metal site. Moreover, the resulting forced electronic structure, at least in certain cases, becomes essential for the protein's biochemical function in electron transport (7). We should remember the entatic hypothesis is in some respects still controversial. Results from some quantum calculations have suggested that the geometry of metal-ligand complexes identified as being rack-induced are not necessarily highly strained (8), whereas other theoretical studies suggest that the rigidity of the protein may in fact be much more significant than initially thought (9).Cupredoxins, a family of electron-transfer metalloproteins, are believed to adopt such a rack-induced state by way of a distorted tetrahedra...

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Characterization and crystal structure of zinc azurin, a by‐product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin

Cited by 147 publications

References 42 publications

Structural studies on Laz, a promiscuous anticancer Neisserial protein

Structural studies on Laz, a promiscuous anticancer Neisserial protein

Engineering type 1 copper sites in proteins

Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin

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