Stabilization of a Cu-binding site by a highly conserved tryptophan residue.

Copper serves as an essential cofactor for nearly all living organisms. There are still many gaps remaining in our knowledge of how Gram-positive bacteria import copper and maintain homeostasis. To obtain a better understanding of how these processes work, here we focus on the ycnKJI operon responsible for regulating copper levels in the Gram-positive bacterium Bacillus subtilis.

Orchestrating copper binding: structure and variations on the cupredoxin fold.

A large number of copper binding proteins coordinate metal ions using a shared three-dimensional fold called the cupredoxin domain. This domain was originally identified in Type 1 "blue copper" centers but has since proven to be a common domain architecture within an increasingly large and diverse group of copper binding domains. The cupredoxin fold has a number of qualities that make it ideal for coordinating Cu ions for purposes including electron transfer, enzyme catalysis, assembly of other copper sites, and copper sequestration.

The YcnI protein from Bacillus subtilis contains a copper-binding domain.

Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram-positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through the ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized Domain of Unknown Function 1775 (DUF1775) containing YcnI.

Crystallographic Studies of the Cerebral Cavernous Malformations Proteins.

Cerebral cavernous malformations (CCM) are dysplasias that primarily occur in the neurovasculature, and are associated with mutations in three genes: KRIT1, CCM2, and PDCD10, the protein products of which are KRIT1 (Krev/Rap1 Interaction Trapped 1; CCM1, cerebral cavernous malformations 1), CCM2 (cerebral cavernous malformations 2; OSM, osmosensing scaffold for MEKK3), and CCM3 (cerebral cavernous malformations 3; PDCD10, programmed cell death 10). Until recently, these proteins were relatively understudied at the molecular level, and only three folded domains were documented. These were a band 4.

PCuC domains from methane-oxidizing bacteria use a histidine brace to bind copper.

Copper is critically important for methanotrophic bacteria because their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is copper-dependent. In addition to pMMO, many other copper proteins are encoded in the genomes of methanotrophs, including proteins that contain eriplasmic opper-haperone (PCuC) domains. Using bioinformatics analyses, we identified three distinct classes of PCuC domain-containing proteins in methanotrophs, termed PmoF1, PmoF2, and PmoF3.

Formation and Electronic Structure of an Atypical Cu Site.

PmoD, a recently discovered protein from methane-oxidizing bacteria, forms a homodimer with a dicopper Cu center at the dimer interface. Although the optical and electron paramagnetic resonance (EPR) spectroscopic signatures of the PmoD Cu bear similarities to those of canonical Cu sites, there are also some puzzling differences. Here we have characterized the rapid formation (seconds) and slow decay (hours) of this homodimeric Cu site to two mononuclear Cu sites, as well as its electronic and geometric structure, using stopped-flow optical and advanced paramagnetic resonance spectroscopies.

Characterization of a long overlooked copper protein from methane- and ammonia-oxidizing bacteria.

Methane-oxidizing microbes catalyze the oxidation of the greenhouse gas methane using the copper-dependent enzyme particulate methane monooxygenase (pMMO). Isolated pMMO exhibits lower activity than whole cells, however, suggesting that additional components may be required. A pMMO homolog, ammonia monooxygenase (AMO), converts ammonia to hydroxylamine in ammonia-oxidizing bacteria (AOB) which produce another potent greenhouse gas, nitrous oxide.

Caspase-1 causes truncation and aggregation of the Parkinson's disease-associated protein α-synuclein.

The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear.

Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.

Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development.

CCM2-CCM3 interaction stabilizes their protein expression and permits endothelial network formation.

Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved "HP1" pocket of the CCM3 focal adhesion targeting (FAT) homology domain.

Structural basis for the disruption of the cerebral cavernous malformations 2 (CCM2) interaction with Krev interaction trapped 1 (KRIT1) by disease-associated mutations.

Familial cerebral cavernous malformations (CCMs) are predominantly neurovascular lesions and are associated with mutations within the KRIT1, CCM2, and PDCD10 genes. The protein products of KRIT1 and CCM2 (Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2), respectively) directly interact with each other. Disease-associated mutations in KRIT1 and CCM2 mostly result in loss of their protein products, although rare missense point mutations can also occur.

Cerebral cavernous malformation proteins at a glance.

Loss-of-function mutations in genes encoding KRIT1 (also known as CCM1), CCM2 (also known as OSM and malcavernin) or PDCD10 (also known as CCM3) cause cerebral cavernous malformations (CCMs). These abnormalities are characterized by dilated leaky blood vessels, especially in the neurovasculature, that result in increased risk of stroke, focal neurological defects and seizures. The three CCM proteins can exist in a trimeric complex, and each of these essential multi-domain adaptor proteins also interacts with a range of signaling, cytoskeletal and adaptor proteins, presumably accounting for their roles in a range of basic cellular processes including cell adhesion, migration, polarity and apoptosis.

Signaling pathways and the cerebral cavernous malformations proteins: lessons from structural biology.

Cerebral cavernous malformations (CCM) are neurovascular dysplasias that result in mulberry-shaped lesions predominantly located in brain and spinal tissues. Mutations in three genes are associated with CCM. These genes encode for the proteins KRIT1/CCM1 (krev interaction trapped 1/cerebral cavernous malformations 1), cerebral cavernous malformations 2, osmosensing scaffold for MEKK3 (CCM2/malcavernin/OSM), and cerebral cavernous malformations 3/programmed cell death 10 (CCM3/PDCD10).

Structural studies of cerebral cavernous malformations 2 (CCM2) reveal a folded helical domain at its C-terminus.

Cerebral cavernous malformations (CCM) are neurovascular dysplasias affecting up to 0.5% of the population. Mutations in the CCM2 gene are associated with acquisition of CCM.