Timothy L. Stemmler

Proteolytic refolding of the HIV‐1 capsid protein amino‐terminus facilitates viral core assembly

After budding, the human immunodeficiency virus (HIV) must ‘mature’ into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix–capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino‐terminal end of the capsid refolds into a β‐hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino‐terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino‐terminus then creates a new CA–CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their amino‐termini formed spheres in vitro, but that removing these residues refolded the capsid amino‐terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA–CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino‐terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti‐ and oncoviruses mature via analogous pathways.

Oxidation of methane by a biological dicopper centre

Vast world reserves of methane gas are underutilized as a feedstock for the production of liquid fuels and chemicals owing to the lack of economical and sustainable strategies for the selective oxidation of methane to methanol. Current processes to activate the strong C–H bond (104 kcal mol-1) in methane require high temperatures, are costly and inefficient, and produce waste. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs thus provide the optimal model for an efficient, environmentally sound catalyst. There are two types of MMO. Soluble MMO (sMMO) is expressed by several strains of methanotroph under copper-limited conditions and oxidizes methane with a well-characterized catalytic di-iron centre. Particulate MMO (pMMO) is an integral membrane metalloenzyme produced by all methanotrophs and is composed of three subunits, pmoA, pmoB and pmoC, arranged in a trimeric α3β3γ3 complex. Despite 20 years of research and the availability of two crystal structures, the metal composition and location of the pMMO metal active site are not known. Here we show that pMMO activity is dependent on copper, not iron, and that the copper active site is located in the soluble domains of the pmoB subunit rather than within the membrane. Recombinant soluble fragments of pmoB (spmoB) bind copper and have propylene and methane oxidation activities. Disruption of each copper centre in spmoB by mutagenesis indicates that the active site is a dicopper centre. These findings help resolve the pMMO controversy and provide a promising new approach to developing environmentally friendly C–H oxidation catalysts.

A Cytosolic Iron Chaperone That Delivers Iron to Ferritin

Ferritins are the main iron storage proteins found in animals, plants, and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. Human poly (rC)–binding protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 can function as a cytosolic iron chaperone in the delivery of iron to ferritin.

Frataxin and Mitochondrial FeS Cluster Biogenesis

Friedreich ataxia is an inherited neurodegenerative disease caused by frataxin deficiency. Frataxin is a conserved mitochondrial protein that plays a role in FeS cluster assembly in mitochondria. FeS clusters are modular cofactors that perform essential functions throughout the cell. They are synthesized by a multistep and multisubunit mitochondrial machinery that includes the scaffold protein Isu for assembling a protein-bound FeS cluster intermediate. Frataxin interacts with Isu, iron, and the cysteine desulfurase Nfs1, which supplies sulfide, thus placing it at the center of mitochondrial FeS cluster biosynthesis.

The Structure and Function of Frataxin

ABSTRACT Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio- and neurodegenerative disorder Friedreichs ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxins specific function remains a point of controversy, the general consensus is that the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the proteins role in different cellular pathways.

The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b.

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.

Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster

Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however. We have purified pMMO from Methylococcus capsulatus (Bath) and detected activity. The purified enzyme has a molecular mass of ≈200 kDa, probably corresponding to an α2β2γ2 polypeptide arrangement. Each 200-kDa pMMO complex contains 4.8 ± 0.8 copper ions and 1.5 ± 0.7 iron ions. Electron paramagnetic resonance spectroscopic parameters corresponding to 40–60% of the total copper are consistent with the presence of a mononuclear type 2 copper site. X-ray absorption near edge spectra indicate that purified pMMO is a mixture of Cu(I) and Cu(II) oxidation states. Finally, extended x-ray absorption fine structure data are best fit with oxygen/nitrogen ligands and a 2.57-Å Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO.

Structure of the Two Transmembrane Cu+ Transport Sites of the Cu+-ATPases

Cu+-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu+ within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu+ transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu+. However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu+-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu+ (Site I Ka = 1.3 fm–1, Site II Ka = 1.1 fm–1), in conjunction with reversible direct Cu+ transfer from chaperones, points to a transport mechanism where backward release of free Cu+ to the cytoplasm is largely prevented.

Crystal Structure and Characterization of Particulate Methane Monooxygenase from Methylocystis species Strain M

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu(I) and Cu(II), of which the latter exhibits two distinct type 2 Cu(II) electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin.

Background: Some iron-dependent enzymes acquire their cofactor from iron chaperones, such as PCBP1. Results: PCBP2, and PCBP3 interact with iron and ferritin in yeast and human cells. Conclusion: All PCBP family members may function as iron chaperones. Significance: The PCBP family members are multifunctional adaptors, mediating interactions between iron or nucleic acids and proteins that act on these molecules. The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C)-binding protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells, and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and -2 exhibited high affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and -4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron-sensitive, ccc1 yeast strain intensified the toxic effects of iron, whereas expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and -2 form a complex for iron delivery to ferritin, and all PCBPs may share iron chaperone activity.