Solution structure and intermolecular interactions of the third metal-binding domain of ATP7A, the Menkes disease protein

The third metal-binding domain of the human Menkes protein (MNK3), a copper(I)-transporting ATPase, has been expressed in Escherichia coli and characterized in solution. The solution structure of MNK3, its copper(I)-binding properties, and its interaction with the physiological partner, HAH1, have been studied. MNK3 is the domain most dissimilar in structure from the other domains of the Menkes protein. This is reflected in a significant rearrangement of the last strand of the four-stranded beta-sheet when compared with the other known homologous proteins or protein domains. MNK3 is also peculiar with respect to its interaction with the copper(I) ion, as it was found to be a comparatively weak binder. Copper(I) transfer from metal-loaded HAH1 was observed experimentally, but the metal distribution was shifted toward binding by HAH1. This is at variance with what is observed for the other Menkes domains.     

Intra- and intermolecular interactions of the catalytic domains of human CD45 protein tyrosine phosphatase

We have investigated protein-protein interaction between distinct domains of the human CD45 cytoplasmic region using a yeast two-hybrid system. Consequently, we have found that the spacer region between two tandem PTP domains specifically interacts with the membrane-distal PTP domain (D2). This interaction is mediated by a stretch of amino acid residues in the carboxyl-terminal half of the spacer region. Although the membrane proximal region does not directly interact with either of the two PTP domains, it appears to function in stabilizing the interaction between the spacer region and D2. We also demonstrate that the interaction between the spacer region and D2 might take place intramolecularly.     

The regulation of catalytic activity of the menkes copper-translocating P-type ATPase. Role of high affinity copper-binding sites

The Menkes protein is a transmembrane copper translocating P-type ATPase. Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency. The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood. We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation. We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model. Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity.     

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide‐binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full‐length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.     

Intra- and intermolecular interactions between intracellular domains of receptor protein-tyrosine phosphatases

The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The generally inactive membrane-distal PTP domains (RPTP-D2s) bind and are proposed to regulate the membrane-proximal PTP domains (RPTP-D1s). We set out to characterize the interactions between RPTP-D1s and RPTP-D2s in vivo by co-immunoprecipitation of hemagglutinin-tagged fusion proteins encoding the transmembrane domain and RPTP-D1 and myc-tagged RPTP-D2. Seven RPTPs from four different subfamilies were used: RPTPalpha, RPTPepsilon, LAR, RPTPvarsigma, RPTPdelta, CD45, and RPTP(mu). We found that RPTP-D2s bound to RPTPs with different affinities. The presence of intrinsic RPTP-D2 altered the binding specificity toward other RPTP-D2s positively or negatively, depending on the identity of the RPTPs. Furthermore, the C terminus of RPTP-D2s and the "wedge" in RPTP-D1s played a central role in binding specificity. Finally, full-length RPTPalpha and LAR heterodimerized in an oxidative stress-dependent manner. Like RPTPalpha-D2, the LAR-D2 conformation was affected by oxidative stress, suggesting a common regulatory mechanism for RPTP complex formation. Taken together, interactions between RPTP-D1s and RPTP-D2s are a common but specific mechanism that is likely to be regulated. The RPTP-D2s and the wedge structures are crucial determinants of binding specificity, thus regulating cross-talk between RPTPs.     

Effects of different sources of copper on Ctr1, ATP7A,ATP7B,MT and DMT1 protein and gene expression in Caco-2 cells

Copper sulfate (CuSO₄), micron copper oxide (micron CuO) and nano copper oxide (nano CuO) at different concentrations were, respectively, added to culture media containing Caco-2 cells and their effects on Ctr1, ATP7A/7B, MT and DMT1 gene expression and protein expression were investigated and compared. The results showed that nano CuO promoted mRNA expression of Ctr1 in Caco-2 cells, and the difference was significant compared with micron CuO and CuSO₄. Nano CuO was more effective in promoting the expression of Ctr1 protein than CuSO₄ and micron CuO at the same concentration. Nano CuO at a concentration of 62.5 μM increased the mRNA expression levels of ATP7A and ATP7B, and the difference was significant compared with CuSO₄. The addition of CuSO₄ and nano CuO to the culture media promoted the expression of ATP7B proteins. CuSO₄ at a concentration of 125 μM increased the mRNA expression level of MT in Caco-2 cells, and the difference was significant compared with nano CuO and micron CuO. Nano CuO at a concentration of 62.5 μM inhibited the mRNA expression of DMT1, and the difference was significant compared with CuSO₄ and micron CuO. Thus, the effects of CuSO₄, micron CuO and nano CuO on the expression of copper transport proteins and the genes encoding these proteins differed considerably. Nano CuO has a different uptake and transport mechanism in Caco-2 cells to those of CuSO₄ and micron CuO.     

Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease

We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B.     

The FP domains of PI31 and Fbxo7 have the same protein fold but very different modes of protein–protein interaction

Fbxo7 and PI31 contain a conserved FP domain that mediates the homo-/hetero-dimerization of the proteins. The PI31 FP domain may also interact with the F-box motif in Fbxo7. The FP domain-mediated protein–protein interactions are important for the functions of Fbxo7 and PI31. The crystal structures of the Fbxo7 and PI31 FP domains were determined previously, showing that a C-terminal helix in the Fbxo7 FP domain was not present in the PI31 FP domain. Here, we determine the crystal structure of the PI31 FP domain using a longer protein construct. The structure is comparable to the Fbxo7 FP domain (including the C-terminal helix), indicating that the two FP domains share the same global fold. However, the FP domains also harbor their own characteristic structural features, mainly in the longest loop (which has a largely fixed conformation due to extensive hydrogen bonding and hydrophobic interactions) and the C-terminal end regions. The crystal structures also reveal fundamental differences in the modes of protein–protein interactions mediated by the two FP domains: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We perform modeling studies to show that the domain-specific structural features may dictate specific modes of inter-domain interactions. We propose that a heterodimeric interaction would be mediated by an αβ interface consisting of the α-helical and β-sheet surfaces of the Fbxo7 and PI31 FP domains, respectively. We also discuss the structural/functional significance of various modes of FP domain-mediated protein–protein interactions.     

The domains of protein S from Myxococcus xanthus: structure, stability and interactions

Protein S from Myxococcus xanthus is a member of the beta gamma-crystallin superfamily. Its N and C-terminal domains (NPS and CPS, respectively) show a high degree of structural similarity and possess the capacity to bind two calcium ions per domain. For NPS, their positions were determined by X-ray diffraction at 1.8 A resolution, making use of molecular replacement with the NMR structure as search model. The overall topology of NPS is found to be practically the same as in complete protein S. In natural protein S, the domains fold independently, with a significant increase in stability and cooperativity of folding in the presence of Ca2+. The recombinant isolated domains are stable monomers which do not show any tendency to combine to "nicked" full-length protein S. In order to investigate the stability and folding of natural protein S and its isolated domains, spectroscopic techniques were applied, measuring the reversible urea and temperature-induced unfolding transitions at varying pH. The increment of Ca2+ to the free energy of stabilization amounts to -10 and -5 kJ/mol for NPS and CPS, respectively. For both NPS and CPS, in the absence and in the presence of 3 mM CaCl2, the two-state model is valid. Comparing DeltaGU-->N for CPS (-21 kJ/mol at pH 7, liganded with Ca2+) with its increment in the intact two-domain protein, the stability of the isolated domain turns out to be decreased in a pH-dependent manner. In contrast, the stability of Ca2+-loaded NPS (DeltaGU-->N=-31 kJ/mol, pH 7) is nearly unchanged down to pH 2 where Ca2+ is released (DeltaGU-->N=-26 kJ/mol, pH 2). In intact protein S, the N-terminal domain is destabilized relative to NPS. Evidently, apart from Ca2+ binding, well-defined domain interactions contribute significantly to the overall stability of intact protein S.     

An atomic-level investigation of the disease-causing A629P mutant of the Menkes protein, ATP7A

Menkes disease is a fatal disease that can be induced by various mutations in the ATP7A gene, leading to unpaired uptake of dietary copper. The ATP7A gene encodes a copper(I)-translocating ATPase. Here the disease-causing A629P mutation, which occurs in the last of the six copper(I)-binding soluble domains of the ATPase (hereafter MNK6), was investigated. To understand why this apparently minor amino acid replacement is pathogenic, the solution structures and dynamics on various time-scales of wild-type and A629P-MNK6 were determined both in the apo- and copper(I)-loaded forms. The interaction in vitro with the physiological ATP7A copper(I)-donor (HAH1) was additionally studied. The A629P mutation makes the protein beta-sheet more solvent accessible, possibly resulting in an enhanced susceptibility of ATP7A to proteolytic cleavage and/or in reduced capability of copper(I)-translocation. A small reduction of the affinity for copper(I) is also observed. Both effects could concur to pathogenicity.     

Trafficking of the copper-ATPases, ATP7A and ATP7B: role in copper homeostasis

Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.     

Copper-transporting ATPases ATP7A and ATP7B: cousins, not twins

Copper plays an essential role in human physiology and is indispensable for normal growth and development. Enzymes that are involved in connective tissue formation, neurotransmitter biosynthesis, iron transport, and others essential physiological processes require copper as a cofactor to mediate their reactions. The biosynthetic incorporation of copper into these enzymes takes places within the secretory pathway and is critically dependent on the activity of copper-transporting ATPases ATP7A or ATP7B. In addition, ATP7A and ATP7B regulate intracellular copper concentration by removing excess copper from the cell. These two transporters belong to the family of P₁-type ATPases, share significant sequence similarity, utilize the same general mechanism for their function, and show partial colocalization in some cells. However, the distinct biochemical characteristics and dissimilar trafficking properties of ATP7A and ATP7B in cells, in which they are co-expressed, indicate that specific functions of these two copper-transporting ATPases are not identical. Immuno-detection studies in cells and tissues have begun to suggest specific roles for ATP7A and ATP7B. These experiments also revealed technical challenges associated with quantitative detection of copper-transporting ATPases in tissues, as illustrated here by comparing the results of ATP7A and ATP7B immunodetection in mouse cerebellum. This work was supported by the National Institute of Health grants PO1 GM 067166–01 and DK R01 DK071865 to S.L.     

Tetramerization and ATP binding by a protein comprising the A, B, and C domains of rat synapsin I

Synapsins are multidomain proteins that are critical for regulating neurotransmitter release in vertebrates. In the present study, two crystal structures of the C domain of rat synapsin I (rSynI-C) in complex with Ca(2+) and ATP reveal that this protein can form a tetramer and that a flexible loop (the "multifunctional loop") contacts bound ATP. Further experiments were carried out on a protein comprising the A, B, and C domains of rat synapsin I (rSynI-ABC). An ATP-stabilized tetramer of rSynI-ABC is observed during velocity sedimentation and size-exclusion chromatographic experiments. These hydrodynamic results also indicate that the A and B domains exist in an extended conformation. Calorimetric measurements of ATP binding to wild-type and mutant rSynI-ABC demonstrate that the multifunctional loop and a cross-tetramer contact are important for ATP binding. The evidence supports a view of synapsin I as an ATP-utilizing, tetrameric protein made up of monomers that have a flexible, extended N terminus.     

A novel copper-binding fold for the periplasmic copper resistance protein CusF

We have determined the crystal structure of apo-CusF, a periplasmic protein involved in copper and silver resistance in Escherichia coli. The protein forms a five-stranded beta-barrel, classified as an OB-fold, which is a unique topology for a copper-binding protein. NMR chemical shift mapping experiments suggest that Cu(I) is bound by conserved residues H36, M47, and M49 located in beta-strands 2 and 3. These residues are clustered at one end of the beta-barrel, and their side chains are oriented toward the interior of the barrel. Cu(I) can be modeled into the apo-CusF structure with only minimal structural changes using H36, M47, and M49 as ligands. The unique structure and metal binding site of CusF are distinct from those of previously characterized copper-binding proteins.     

A soluble C1b protein and its regulation of soluble type 7 adenylyl cyclase

Adenylyl cyclase (AC) is a prototypical cell-signaling molecule expressed in virtually all organisms from bacteria to man. While C1b, a poorly conserved region within mammalian AC, has been implicated in numerous isoform-specific regulatory properties, no one has purified the C1b region as a functional protein to homogeneity in order to study its role in enzyme function. We hypothesize that C1b is an internal regulatory subunit. To pursue this hypothesis, we constructed several soluble C1b proteins from type VII AC, arriving at one, 7C1b-S, which can be expressed and purified from Escherichia coli. 7C1b-S is relatively stable, as demonstrated by limited proteolytic analysis, circular dichroism, and UV Raman spectroscopy. Using size-exclusion chromatography and co-immunoprecipitation we demonstrate that 7C1b-S interacts with a cardinal activator of AC (Gsalpha) and with the conserved first catalytic domain (C1a) of type VII AC. We show that 7C1b-S inhibits Gsalpha-stimulated and Gsalpha-forskolin stimulated activity in our soluble ACVII model system. On the basis of these results, we suggest that 7C1b-S meets basic criteria to serve as a model protein for the C1b region and may be used as a prototype to develop other isoform C1b soluble model proteins to further investigate the role of this domain in isoform-specific regulation of adenylyl cyclase.     

Identifying protein domains by global analysis of soluble fragment data

The production and analysis of individual structural domains is a common strategy for studying large or complex proteins, which may be experimentally intractable in their full-length form. However, identifying domain boundaries is challenging if there is little structural information concerning the protein target. One experimental procedure for mapping domains is to screen a library of random protein fragments for solubility, since truncation of a domain will typically expose hydrophobic groups, leading to poor fragment solubility. We have coupled fragment solubility screening with global data analysis to develop an effective method for identifying structural domains within a protein. A gene fragment library is generated using mechanical shearing, or by uracil doping of the gene and a uracil-specific enzymatic digest. A split green fluorescent protein (GFP) assay is used to screen the corresponding protein fragments for solubility when expressed in Escherichia coli. The soluble fragment data are then analyzed using two complementary approaches. Fragmentation “hotspots” indicate possible interdomain regions. Clustering algorithms are used to group related fragments, and concomitantly predict domain location. The effectiveness of this Domain Seeking procedure is demonstrated by application to the well-characterized human protein p85α.     

Experimental mapping of soluble protein domains using a hierarchical approach

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.