Unique structural organization of ATP-dependent LonA proteases

Homooligomeric LonA proteases are the key components of the protein quality control system in bacteria and eukaryotes. Domain organization of the common pool of LonA proteases is determined by the comparative analysis of primary and secondary structures of a number of bacterial and eukaryotic enzymes. The similarity of individual enzyme domains was estimated, domain-domain linker areas were revealed, and regions that are capable of including intercalated peptide fragments were identified. LonA proteases were shown to be unique AAA⁺ proteins, because in addition to the classic AAA⁺ module they contain a part of another AAA⁺ module, namely the α-helical domain including a coiled-coil region, which is similar to the α-helical domain of the AAA⁺-1 module of the chaperone-disagregases ClpB/Hsp104.     

The AAA motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71 kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA⁺ superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA⁺ class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.     

The regulatory function of N-terminal AAA+ ATPase domain of eukaryote-like archaeal Orc1/Cdc6 protein during DNA replication initiation

Archaeal replication machinery represents a core version of this in eukaryotes. The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). In this study, we investigate the DNA-binding activities of the N-terminal AAA⁺ ATPase domains of these Orc1/Cdc6 proteins, including their functional interactions with the other SsoCdc6 proteins, on duplex DNA substrates derived from the origins of S. solfataricus. We showed that the ATPase domain of SsoCdc6-2 retained to a great extent the origin DNA-binding activity, and likewise maintained its stimulating effect on SsoCdc6-3. Second, the ATPase domain of SsoCdc6-1, which also stimulated the DNA-binding ability of SsoCdc6-3, demonstrated a significantly improved DNA-binding activity at the forked substrate, but only showed a very weak ability towards the blunt DNA. Third, the ATPase domain of SsoCdc6-3, although having lost much of its DNA-binding activity from the origin, inhibited both SsoCdc6-1 and SsoCdc6-2. These imply that the N-terminal AAA⁺ ATPase domain of archaeal Orc1/Cdc6 protein could be differentially involved in origin recognition during DNA replication initiation even if lacking conventional C-terminal winged helix DNA-binding elements. Our findings further propose that conserved AAA⁺ ATPase domains of Orc1/Cdc6 proteins determine their defined and coordinated functions not only in the archaeon species but also in eukaryotes during the early events of DNA replication.     

Comparative Study of the Action of Bovine Duodenal Proteinases (Duodenases) on Polypeptide Substrates

A comparative study of substrate specificity of bovine duodenal proteinases—chymotrypsin-like duodenase (ChlD) and dual-specificity duodenase (dsD)—was carried out using oligopeptide substrates (human proinsulin, glucagon, melittin, angiotensinogen fragment 1-14). ChlD displayed mainly chymotrypsin-like properties towards these substrates, hydrolyzing peptide bonds carboxy-terminally to bulky aliphatic or aromatic residues. In melittin, ChlD additionally cleaved peptide bonds after Thr and Ser residues. Dual-specificity duodenase (dsD) significantly restricted its specificity to only trypsin-like or only chymotrypsin-like or displayed full activity, combining both specificities, depending on substrate. Both ChlD and dsD efficiently hydrolyzed a single peptide bond (Phe8–His9) in angiotensinogen fragment 1-14. The kinetic parameters of angiotensinogen fragment 1-14 cleavage by ChlD and dsD were determined (k _cat/K _m = 80,500 M^-1·sec^-1 for ChlD and 103,000 M^-1·sec^-1 for dsD).     

Novel essential residues of Hda for interaction with DnaA in the regulatory inactivation of DnaA: unique roles for Hda AAA+ Box VI and VII motifs

Escherichia coli ATP–DnaA initiates chromosomal replication. For preventing extra‐initiations, a complex of ADP–Hda and the DNA‐loaded replicase clamp promotes DnaA‐ATP hydrolysis, yielding inactive ADP–DnaA. However, the Hda–DnaA interaction mode remains unclear except that the Hda Box VII Arg finger (Arg‐153) and DnaA sensor II Arg‐334 within each AAA⁺ domain are crucial for the DnaA‐ATP hydrolysis. Here, we demonstrate that direct and functional interaction of ADP–Hda with DnaA requires the Hda residues Ser‐152, Phe‐118 and Asn‐122 as well as Hda Arg‐153 and DnaA Arg‐334. Structural analyses suggest intermolecular interactions between Hda Ser‐152 and DnaA Arg‐334 and between Hda Phe‐118 and the DnaA Walker B motif region, in addition to an intramolecular interaction between Hda Asn‐122 and Arg‐153. These interactions likely sustain a specific association of ADP–Hda and DnaA, promoting DnaA‐ATP hydrolysis. Consistently, ATP–DnaA and ADP–DnaA interact with the ADP–Hda‐DNA–clamp complex with similar affinities. Hda Phe‐118 and Asn‐122 are contained in the Box VI region, and their hydrophobic and electrostatic features are basically conserved in the corresponding residues of other AAA⁺ proteins, suggesting a conserved role for Box VI. These findings indicate novel interaction mechanisms for Hda–DnaA as well as a potentially fundamental mechanism in AAA⁺ protein interactions.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Trapping of a transcription complex using a new nucleotide analogue: AMP aluminium fluoride

Mechanochemical proteins rely on ATP hydrolysis to establish the different functional states required for their biological output. Studying the transient functional intermediate states these proteins adopt as they progress through the ATP hydrolysis cycle is key to understanding the molecular basis of their mechanism. Many of these intermediates have been successfully ‘trapped’ and functionally characterised using ATP analogues. Here, we present a new nucleotide analogue, AMP-AlF_x, which traps PspF, a bacterial enhancer binding protein, in a stable complex with the σ⁵⁴-RNA polymerase holoenzyme. The crystal structure of AMP-AlF_x•PspF_1-275 provides new information on protein-nucleotide interactions and suggests that the β and γ phosphates are more important than the α phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP-AlF_x establish distinct roles for the conserved catalytic AAA⁺ (ATPases associated with various cellular activities) residues, suggesting that AMP-AlF_x is a powerful new tool to study AAA⁺ protein family members and, more generally, Walker motif ATPases.     

Modulation of TRPM6 and Na+/Mg2+ exchange in mammary epithelial cells in response to variations of magnesium availability

Mammary epithelial cells (HC11) chronically adapted to grow in a low‐magnesium (0.05 mM vs. 0.5 mM) or in a high‐magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non‐physiological magnesium availability. Magnesium influx was higher in low‐magnesium cells compared to control or high‐magnesium cells, whereas magnesium efflux was higher in high‐magnesium cells compared to control and low‐magnesium cells. Magnesium efflux was partially inhibited by imipramine, inhibitor of the Na⁺/Mg²⁺ exchange. Using a monoclonal antibody detecting a ～70 kDa protein associated with Na⁺/Mg²⁺ exchange activity, we found that the expression levels of this protein were proportional to magnesium efflux capacity, that is, high‐magnesium cells > control cells > low‐magnesium cells. As for magnesium influx, this was abolished by Co(III)hexaammine, inhibitor of magnesium channels. Surprisingly, we found that cells grown in low magnesium upregulated mRNA for the magnesium channel TRPM6, but not for other channels like TRPM7 or MagT1. TRPM6 mRNA was also rapidly upregulated or downregulated in HC11 cells deprived of magnesium or in low‐magnesium cells re‐added with magnesium, respectively. TRPM6 protein levels, as assessed by Western blot and immunofluorescence, underwent similar changes under comparable conditions. We propose that mammary epithelial cells adapt to decreased magnesium availability by upregulating magnesium influx via TRPM6, and counteract increased magnesium availability by increasing magnesium efflux primarily via Na⁺/Mg²⁺ exchange. These results show, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells, similar to what is known for intestine and kidney. J. Cell. Physiol. 222: 374–381, 2010. © 2009 Wiley‐Liss, Inc.     

A novel view on the architecture of the non-catalytic N-terminal region of ATP-dependent LonA proteases

ATP-Dependent Lon-proteases are components of the protein quality control system, which maintains cellular proteome. The Lon family consists of two subfamilies A and B, differing in subunit architecture and intracellular location. We propose here a reinterpretation of the domain organization of the non-catalytic N-terminal region of LonA proteases. Using Escherichia coli LonA protease (EcLon) as an example, it has been shown that a fragment (αN domain) located between the N-terminal domain and the AAA⁺ module is similar to the α1 domain of the first AAA⁺ module of chaperone-disaggregase ClpB. A coiled-coil (CC) region included in the αN domain of LonA is similar to the M domain of ClpB chaperones, which is inserted into the α1 domain. This region is suggested to adopt the structure similar to the propeller-like (PL) domain. The typical architecture of the N-terminal region of LonA proteases is postulated to be characterized by the obligatory presence of a PL domain, included in the αN domain, but may vary in the length and topology of the preceding N-terminal domain, which can have in some cases a more complex structure than in EcLon.     

Loss of spindle assembly checkpoint–mediated inhibition of Cdc20 promotes tumorigenesis in mice

Genomic instability is a hallmark of human cancers. Spindle assembly checkpoint (SAC) is a critical cellular mechanism that prevents chromosome missegregation and therefore aneuploidy by blocking premature separation of sister chromatids. Thus, SAC, much like the DNA damage checkpoint, is essential for genome stability. In this study, we report the generation and analysis of mice carrying a Cdc20 allele in which three residues critical for the interaction with Mad2 were mutated to alanine. The mutant Cdc20 protein (AAA-Cdc20) is no longer inhibited by Mad2 in response to SAC activation, leading to the dysfunction of SAC and aneuploidy. The dysfunction could not be rescued by the additional expression of another Cdc20 inhibitor, BubR1. Furthermore, we found that Cdc20^AAA/AAA mice died at late gestation, but Cdc20^+/AAA mice were viable. Importantly, Cdc20^+/AAA mice developed spontaneous tumors at highly accelerated rates, indicating that the SAC-mediated inhibition of Cdc20 is an important tumor-suppressing mechanism.     

The structure and regulation of magnesium selective ion channels

The magnesium ion (Mg^2 +) is the most abundant divalent cation within cells. In man, Mg^2 +-deficiency is associated with diseases affecting the heart, muscle, bone, immune, and nervous systems. Despite its impact on human health, little is known about the molecular mechanisms that regulate magnesium transport and storage. Complete structural information on eukaryotic Mg^2 +-transport proteins is currently lacking due to associated technical challenges. The prokaryotic MgtE and CorA magnesium transport systems have recently succumbed to structure determination by X-ray crystallography, providing first views of these ubiquitous and essential Mg^2 +-channels. MgtE and CorA are unique among known membrane protein structures, each revealing a novel protein fold containing distinct arrangements of ten transmembrane-spanning α-helices. Structural and functional analyses have established that Mg^2 +-selectivity in MgtE and CorA occurs through distinct mechanisms. Conserved acidic side-chains appear to form the selectivity filter in MgtE, whereas conserved asparagines coordinate hydrated Mg^2 +-ions within the selectivity filter of CorA. Common structural themes have also emerged whereby MgtE and CorA sense and respond to physiologically relevant, intracellular Mg^2 +-levels through dedicated regulatory domains. Within these domains, multiple primary and secondary Mg^2 +-binding sites serve to staple these ion channels into their respective closed conformations, implying that Mg^2 +-transport is well guarded and very tightly regulated. The MgtE and CorA proteins represent valuable structural templates to better understand the related eukaryotic SLC41 and Mrs2–Alr1 magnesium channels. Herein, we review the structure, function and regulation of MgtE and CorA and consider these unique proteins within the expanding universe of ion channel and transporter structural biology.     

Asymmetric nucleotide transactions of the HslUV protease

ATP binding and hydrolysis are critical for protein degradation by HslUV, a AAA ⁺ machine containing one or two HslU₆ ATPases and the HslV₁₂ peptidase. Although each HslU homohexamer has six potential ATP-binding sites, we show that only three or four ATP molecules bind at saturation and present evidence for three functional subunit classes. These results imply that only a subset of HslU and HslUV crystal structures represents functional enzyme conformations. Our results support an asymmetric mechanism of ATP binding and hydrolysis, and suggest that molecular contacts between HslU and HslV vary dynamically throughout the ATPase cycle. Nucleotide binding controls HslUV assembly and activity. Binding of a single ATP allows HslU to bind HslV, whereas additional ATPs must bind HslU to support substrate recognition and to activate ATP hydrolysis, which powers substrate unfolding and translocation. Thus, a simple thermodynamic hierarchy ensures that substrates bind to functional HslUV complexes, that ATP hydrolysis is efficiently coupled to protein degradation, and that working HslUV does not dissociate, allowing highly processive degradation.     

Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

VPS4 proteins are AAA⁺ ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA⁺ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His₆-VPS4A on liposomes containing Ni²⁺-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.     

Subunit organization of Mcm2-7 and the unequal role of active sites in ATP hydrolysis and viability

The Mcm2-7 (minichromosome maintenance) complex is a toroidal AAA⁺ ATPase and the putative eukaryotic replicative helicase. Unlike a typical homohexameric helicase, Mcm2-7 contains six distinct, essential, and evolutionarily conserved subunits. Precedence to other AAA⁺ proteins suggests that Mcm ATPase active sites are formed combinatorially, with Walker A and B motifs contributed by one subunit and a catalytically essential arginine (arginine finger) contributed by the adjacent subunit. To test this prediction, we used copurification experiments to identify five distinct and stable Mcm dimer combinations as potential active sites; these subunit associations predict the architecture of the Mcm2-7 complex. Through the use of mutant subunits, we establish that at least three sites are active for ATP hydrolysis and have a canonical AAA⁺ configuration. In isolation, these five active-site dimers have a wide range of ATPase activities. Using Walker B and arginine finger mutations in defined Mcm subunits, we demonstrate that these sites similarly make differential contributions toward viability and ATP hydrolysis within the intact hexamer. Our conclusions predict a structural discontinuity between Mcm2 and Mcm5 and demonstrate that in contrast to other hexameric helicases, the six Mcm2-7 active sites are functionally distinct.     

The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis

Clathrin‐mediated endocytosis is a fundamental transport pathway that depends on numerous protein‐protein interactions. Testing the importance of the adaptor protein‐clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin‐binding motif (sla1^AAA) that disrupt clathrin binding. Live‐cell imaging showed an impaired Sla1‐clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1^AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3‐dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1^AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1‐clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.      

SUMOylation alters CRMP2 regulation of calcium influx in sensory neurons

The axon/dendrite specification collapsin response mediator protein 2 (CRMP2) bidirectionally modulates N-type voltage-gated Ca²⁺ channels (CaV2.2). Here we demonstrate that small ubiquitin-like modifier (SUMO) protein modifies CRMP2 via the SUMO E2-conjugating enzyme Ubc9 in vivo. Removal of a SUMO conjugation site KMD in CRMP2 (K374A/M375A/D376A; CRMP2_AAA) resulted in loss of SUMOylated CRMP2 without compromising neurite branching, a canonical hallmark of CRMP2 function. Increasing SUMOylation levels correlated inversely with calcium influx in sensory neurons. CRMP2 deSUMOylation by SUMO proteases SENP1 and SENP2 normalized calcium influx to those in the CRMP2_AAA mutant. Thus, our results identify a novel role for SUMO modification in CRMP2/CaV2.2 signaling pathway.