Crystal structures of <i>E. coli</i> CcmG and its mutants reveal key roles of the N‐terminal β‐sheet and the fingerprint region

Ouyang, Nan; Gao, Yong-Guang; Hu, Hong Yu; Xia, Zeyang

doi:10.1002/prot.21184

Cited by 27 publications

(30 citation statements)

References 41 publications

(52 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These observations are consistent with the instability of a soluble version of the P141S variant and suggest that Pro141 is important for the stability of the protein fold. This contrasts with a report for E. coli CcmG, in which replacement of the cisproline, P144, by an alanine resulted in a stable protein with an overall structure very similar to that of the wild-type protein (but with altered redox potential properties) (31).…”

Section: Replacement Of the N-terminal Transmembrane Anchor Ofcontrasting

confidence: 91%

The Active-Site Cysteinyls and Hydrophobic Cavity Residues of ResA Are Important for CytochromecMaturation inBacillus subtilis

et al. 2008

View full text Add to dashboard Cite

ResA is an extracytoplasmic membrane-bound thiol-disulfide oxidoreductase required for cytochrome c maturation in Bacillus subtilis. Previous biochemical and structural studies have revealed that the active-site cysteinyls cycle between oxidized and reduced states with a low reduction potential and that, upon reduction, a hydrophobic cavity forms close to the active site. Here we report in vivo studies of ResA-deficient B. subtilis complemented with a series of ResA variants. Using a range of methods to analyze the cellular cytochrome c content, we demonstrated (i) that the N-terminal transmembrane segment of ResA serves principally to anchor the protein to the cytoplasmic membrane but also plays a role in mediating the activity of the protein; (ii) that the active-site cysteines are important for cytochrome c maturation activity; (iii) that Pro141, which forms part of the hydrophobic cavity and which adopts a cis conformation, plays an important role in protein stability; (iv) that Glu80, which lies at the base of the hydrophobic cavity, is important for cytochrome c maturation activity; and, finally, (v) that Pro141 and Glu80 ResA mutant variants promote selective maturation of low levels of one c-type cytochrome, subunit II of the cytochrome c oxidase caa 3 , indicating that this apocytochrome is distinct from the other three endogenous c-type cytochromes of B. subtilis.

show abstract

Section: Replacement Of the N-terminal Transmembrane Anchor Ofcontrasting

confidence: 91%

The Active-Site Cysteinyls and Hydrophobic Cavity Residues of ResA Are Important for CytochromecMaturation inBacillus subtilis

et al. 2008

View full text Add to dashboard Cite

show abstract

“…By way of comparison, it is known that even relatively minor changes in the heme pocket structure can have significant effects on the stability of oxy-heme. For example, an Mb mutant with the side chains of His 64 and Val 68 interchanged adopts a bis-histidyl heme coordination geometry and undergoes very rapid autoxidation in the presence of oxygen (45). The current study suggests one mechanism by which AHSP may destabilize the oxy-␣Hb structure.…”

Section: Discussionmentioning

confidence: 99%

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

Gell

Feng

Zhou

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

␣-Hemoglobin (␣Hb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with ␣Hb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting ␣Hb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in ␣Hb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro 30 , in loop 1 of AHSP. Mutation of Pro 30 to a variety of residue types results in reduced ability to convert ␣Hb. In complex with ␣Hb, AHSP Pro 30 adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in ␣Hb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the ␣Hb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of ␣Hb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.Mammalian adult hemoglobin (HbA) 5 is a tetramer of two ␣Hb and two ␤Hb subunits, which is produced to extremely high concentrations (ϳ340 mg/ml) in red blood cells. Numerous mechanisms exist to balance and coordinate HbA synthesis in normal erythropoiesis, and problems with the production of either HbA subunit give rise to thalassemia, a common cause of anemia worldwide. Previously, we identified ␣-hemoglobin stabilizing protein (AHSP) as an accessory factor in normal HbA production (1). AHSP forms a dimeric complex with ␣Hb (see Fig. 1A) (2) but does not interact with ␤Hb or HbA. AHSP also binds heme-free (apo) ␣Hb (3) and may serve functions in both the folding of nascent ␣Hb (4) and the detoxification of excess ␣Hb that remains following HbA assembly (2, 5). Mice carrying an Ahsp gene knock-out display mild anemia, ineffective erythropoiesis, and enhanced sensitivity to oxidative stress (1, 6), features also observed in ␤-thalassemia patients due to the cytotoxic effects of free ␣Hb.Free ␣Hb promotes the formation of harmful reactive oxygen species as a result of reduction/oxidation reactions involving the heme iron (7,8). Reactive oxygen species can damage heme, ␣Hb, and other cellular structures, resulting in hemoglobin precipitates and death of erythroid precursor cells (9 -12). The presence of AHSP may explain how cells tolerate the slight excess of ␣Hb that is observed in normal erythropoiesis, which is postulated to inhibit the formation of non-functional ␤Hb tetramers, thus providing a robust mechanism for achieving the correct subunit stoichiometry during HbA assembly (13).Structural and biochemical studies have begun to elucidate the molecular mechanism by which AHSP detoxifies ␣Hb. AHSP binds to oxygenated ␣Hb to generate an initial complex that retains the oxy-hem...

show abstract

“…3A) can be described as a modified thioredoxin-like fold that is highly reminiscent of B. subtilis ResA (12), CcmG (from E. coli and Bradyrhizobium japonicum) (26,27), and Mycobacterium tuberculosis DsbE (28), which are all extracytoplasmic TDORs (see Fig. 3B).…”

Section: Resultsmentioning

confidence: 99%

Structure and Functional Properties of Bacillus subtilis Endospore Biogenesis Factor StoA

Crow

Liu

Möller

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Bacillus subtilisStoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxinlike fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as ؊248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pK a values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but nonredundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.

show abstract

Crystal structures of E. coli CcmG and its mutants reveal key roles of the N‐terminal β‐sheet and the fingerprint region

Cited by 27 publications

References 41 publications

The Active-Site Cysteinyls and Hydrophobic Cavity Residues of ResA Are Important for CytochromecMaturation inBacillus subtilis

The Active-Site Cysteinyls and Hydrophobic Cavity Residues of ResA Are Important for CytochromecMaturation inBacillus subtilis

A cis-Proline in α-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of α-Hemoglobin

Structure and Functional Properties of Bacillus subtilis Endospore Biogenesis Factor StoA

Contact Info

Product

Resources

About