Biochemical Journal

In enzymology, hysteresis is manifested as a time-dependent shift in the kinetic behavior of an enzyme. Through hysteresis, the activation or inhibition of a biological pathway can be regulated by a molecule or metabolite that acts as a hysteretic modulator of the enzyme within that metabolic route. This mechanism of regulation contrasts with those that act on gene expression leading to modulation of enzyme protein levels. Through hysteresis, the amplitude of natural oscillations in metabolic pathways can be adjusted according to the levels of a metabolite that might be beneficial for cells. At physiological level, the slow response of hysteretic enzymes to changes, in the cellular levels of substrates, allows a time-dependent buffering effect on certain metabolites. Understanding the mechanisms and properties of hysteretic enzymes has been important for developing new therapies and improving our understanding of these enzymes in biological systems. However, due to their complex kinetics, the study of hysteretic enzymes has remained a challenge over time. In this study, we characterized the reduction of cytochrome b5 by NADH-dependent microsomal enzymes from rat liver using recombinant purified cytochrome b5, coenzyme Q10 and coenzyme Q0, as substrates, to mimic the conditions found in biological membranes, where competition between cytochrome b5 and other substrates might influence their reduction. We found a lag-time-dependent behavior in the cytochrome b5 reduction compatible with the existence of hysteretic modulation induced by endogenous molecules present in these membranes. Our data suggest that at least for the case of coenzyme Q10, fluctuations in its levels may impact metabolic pathways in which reduced cytochrome b5 levels play a key for the function of the cytochrome b5-dependent route.

Collagen triple helix folds in two steps: nucleation of three polypeptides at the C-termini followed by zip-chain like propagation. The triple helices found in all domains of life as well as viruses contain upto 6000 amino acids in each polypeptide that are also frequently interrupted with non-helical sequences that disrupt folding and reduce stability. Given the length of polypeptide and the disruptive interruptions, compensating mechanisms that stabilize against local unfolding during propagation and offset the entropic cost of folding the long polypeptides are not fully understood. Here, we show that the information for correct folding of collagen triple helices is encoded in their sequence as interchain electrostatic interactions. In case of humans, disrupting these interactions causes severe to lethal diseases. Key ResultCollagen triple helices found in all the three domains of life as well as viruses have converged on similar mechanism to fold correctly.

Mitochondrial creatine kinases are key players in maintaining energy homeostasis in cells by working in conjunction with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. High levels of MtCK observed in Her2+ breast cancer and inhibition of breast cancer cell growth by substrate analog, cyclocreatine, indicate dependence of cancer cells on the energy shuttle for cell growth and survival. Hence, understanding the key mechanistic features of creatine kinases and their inhibition plays an important role in the development of cancer therapeutics. Herein, we present the mutational and structural investigation on understudied ubiquitous mitochondrial creatine kinase (uMtCK). Our cryo-EM structures and biochemical data on uMtCK showed closure of the loop comprising residue His61 is specific to and relies on creatine binding and the reaction mechanism of phosphoryl transfer depends on electrostatics in the active site. In addition, the previously identified covalent inhibitor CKi showed inhibition in breast cancer BT474 cells, however our biochemical and structural data indicated that CKi is not a potent inhibitor for breast cancer due to strong dependency on the covalent link formation and inability to induce conformational changes upon binding.

Disulfide bond formation is crucial to the structure and function of many proteins. It is known that there is diversity in the pathways for disulfide bond formation in bacteria and that there are gaps in our knowledge of these pathways. Using a combination of experimental and bioinformatic approaches we show that some of these gaps can be filled by a newly discovered oxidative folding pathway centered on methylamine utilization protein E (MauE). MauE has previously been associated with the methylamine utilization (MAU) gene cluster, which is involved in methylamine metabolism, in particular it is associated with the maturation of the small subunit of methylamine dehydrogenase. Here we show MauE from Caldithrix abyssi and Desulfatibacillum alphaticivorans functionally replace disulfide bond formation protein B (DsbB) in E. coli using two independent disulfide bond dependent assays. Furthermore, MauE is found in 14 species from 2 bacterial phyla that lack known pathways for structural disulfide bond formation, but which have proteins with structural disulfide bonds in the protein data bank. The active site for MauE was determined to be a conserved CXC motif. Using molecular docking predictions, we demonstrate that MauE is likely to interact with ubiquinone, similarly to the well characterized bacterial DsbB. We also constructed a dataset across thirty-five different phyla to demonstrate that MauE is potentially the second most common disulfide bond formation protein in bacterial disulfide bond formation pathways after DsbB. In addition, the distribution of MauE largely differs from the distribution of other MAU gene cluster markers affirming its role as a newly discovered generalist disulfide bond formation protein rather than being a specialized maturation factor for methylamine dehydrogenase. We also reveal further gaps in disulfide bond pathways, as well as species which may contain redundancies in their disulfide bond pathways.

External stress can disrupt protein homeostasis in organisms, necessitating the involvement of heat shock proteins (Hsps) to restore equilibrium and ensure survival. Unlike other organisms, the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius lacks Hsp100, Hsp90, and Hsp70, possessing only two small heat shock proteins (Hsp14 and Hsp20) and one group II chaperonin, Hsp60. This raises questions about how protein substrates are protected and transferred to Hsp60 for refolding without other chaperones. Our study focused on ATP-dependent Hsp60 in S. acidocaldarius, revealing its formation of oligomeric structures in the presence of ATP. While ATP hydrolysis is not essential for oligomer formation and lid closure, it is crucial for Hsp60s chaperone activity, effectively folding stress-denatured substrate proteins by stabilizing their folded conformations. The mechanism involves hydrophobic recognition of unfolded substrates, encapsulating and releasing them in a more folded state. Negatively charged inner surface of the ring seems to be responsible for driving the folding of the substrate. Importantly, Hsp14 was found to transfer substrate proteins to Hsp60{beta}, orchestrating their refolding into an active state. Beyond protein folding, Hsp60{beta} protects the membrane under stress, contributing to maintaining membrane rigidity. Hsp60 exhibits nested cooperativity in ATPase activity, adapting to ATP concentration changes and interestingly Hsp60{beta} and Hsp60{beta} complex shows a mosaic behaviour during ATP hydrolysis belonging to both group I and group II chaperonin respectively. In conclusion, our study provides insights into the intricate mechanisms employed by Hsp60 in S. acidocaldarius to maintain protein homeostasis. It offers a comprehensive understanding of Hsp60s role in the heat shock response pathway, shedding light on fundamental cellular processes in extremophilic archaea.

Archetypal metalloenzyme Cytochrome P450cam (CYP101A1) catalyzes regioselective hydroxylation of its native substrate camphor in heme active site. However, the proposal of potential existence of additional substrate binding modes distal from the active site in P450cam and their concomitant roles in regulating recognition at active site have remained a matter of recurring discourse. Herein we report the discovery of a novel 3site state in P450cam, where three substrate molecules were observed to simultaneously bind to P450cam at three distinct sites including the heme active site. These three binding modes, hereby referred as catalytic, waiting and allosteric binding modes in 3site state, are allosterically inter-linked and function in mutually synergistic fashion. The 3site state possesses regio-selective conformations of substrate essential for catalysis and establishes substrate-ingress and product exit process to and from the active site via two distinct channels. The ensemble of three-state binding modes are found to be self-consistent with NMR pseudo-contact shift data obtained from TROSY-HSQC measurements and DEER based predictions. Binding of redox partner Putidaredoxin with 3site model retains closed conformation of 3site state, siding with NMR based hypothesis that the catalysis would take place in closed insulation of P450cam even in presence of its redox partner. Statement of SignificanceUbiquitous superfamily of mono-oxygenases cytochrome P450s are involved in broad range of metabolic process in all domains of life and are also important drug targets. Apart from the well known and established binding mode in heme active site, the substrate bindings at additional distal sites have been postulated in multitude of P450s. Using the archetypal bacterial cytochrome P450 i.e., P450cam, a novel 3site state of cytochrome P450 is elucidated in this work. The novel 3site state has two additional binding modes namely waiting and allosteric (also postulated previously), apart from known binding mode catalytic in the active site. The known functions of P450cam are found to be most optimally explained by this 3site state, instead of single substrate bound catalytic state. This state can be of critical importance for CYP superfamily at large and potentially be useful in understanding the non-Michaelis behaviour, observed in many P450s.

The insulin superfamily proteins (ISPs), in particular, insulin, IGFs and relaxins are key modulators of animal physiology. They are known to have evolved from the same ancestral gene and have diverged into proteins with varied sequences and distinct functions, but maintain a similar structural architecture stabilized by highly conserved disulphide bridges. A recent surge of sequence data and the structures of these proteins prompted a need for a comprehensive analysis which connects the evolution of these sequences in the light of available functional and structural information and their interaction with cognate receptors. This study reveals a) unusually high sequence conservation of IGFs (>90%), which has never been reported before. In fact, it was interesting to observe that the functional domains (excluding signal peptide) of human, horse, pig and Ords kangaroo rat are 100% identical. (b) an updated definition of the signature motif of the relaxin family (c) a non-canonical C-peptide cleavage site in a few killifish insulin sequences and so on. We also provide a structure-based rationale for such conservation by introducing a concept called binding partners imposed evolutionary constraints. Furthermore, the high conservation of IGFs appears to represent a classic case of resistance to sequence diversity exerted by physiologically important interactions with multiple partners. Furthermore, we propose a probable mechanism for C-peptide cleavage in killifish insulin sequences.

Changes in biochemical properties, caused by iterative mutations in amino acid sequence, underlie the alterations in protein function over time that underpin the evolutionary process. An example is the switching of an enzymes reliance from one essential metal to an alternative as their catalytic cofactor. We previously described such a neofunctionalisation in Staphylococcus aureus, which altered a superoxide dismutase (SOD) enzyme from being an ancestral manganese-dependent (MnSOD) into an extant isozyme that can equally utilise either manganese or iron, termed cambialism (camSOD). Yet its unclear whether camSOD emergence involved selection solely for cofactor flexibility or whether other biochemical properties also diverged during neofunctionalisation. Here, we have investigated an independent biochemical property of the S. aureus SODs, their structural stability. We demonstrate that the neofunctionalised camSOD exhibits increased stability relative to the ancestral MnSOD. S. aureus camSOD is more resistant to both chemical and thermal unfolding in vitro. Crucially, while both isozymes possess a stable core at the heart of their fold, consisting of regions of the protein localised around the metal cofactor that resist hydrogen-deuterium exchange when exposed to isotopically labelled solvent, this core is larger and more exchange-resistant in camSOD than MnSOD. Thus, during the recent divergence of this SOD pair, two distinct biochemical properties have undergone substantial and rapid evolutionary change. This study paves the way for investigations of the structural and functional relationship between these properties, a SODs metal-preference and stability, and of how these properties were concomitantly selected during neofunctionalisation in the S. aureus lineage.

Cryptochromes and photolyases are blue-light-sensitive flavoproteins that generally bind flavin adenine dinucleotide (FAD) and have distinct functions. Cryptochrome 4a (CRY4a) is a protein expressed in the double-cone photoreceptors of the retina in migratory songbirds like European robin (Erithacus rubecula) and is hypothesized as the primary sensor for avian magnetoreception. In addition to FAD, most photolyases and some cryptochromes bind antenna chromophores such as 8-hydroxy-5-deazaflavin (8-HDF) or 5,10-methenyltetrahydrofolate (MTHF) to enhance light absorption. Here, we investigated whether Erithacus rubecula Cryptochrome 4a (ErCRY4a) also binds 8-HDF and/or MTHF. 8-HDF binding was studied by co-expressing ErCRY4a with the fbIC gene that encodes for 8-HDF synthase and thus for production of 8-HDF in E. coli. As a positive control for 8-HDF binding, we expressed Xenopus laevis 6-4 photolyase (Xl6-4PL) which is known to bind both FAD and 8-HDF. This experiment resulted in successful binding of 8-HDF to Xl6-4PL, but not to ErCRY4a. We studied the binding of MTHF using in vitro reconstitution followed by UV-Vis spectroscopy and isothermal titration calorimetry (ITC) assays. No interaction was observed between MTHF and ErCRY4a. To theoretically understand the binding of potential antenna chromophores to ErCRY4a, we performed computational analyses. We found no similarity at the relevant binding sites between the sequences of ErCRY4a with proteins shown to bind MTHF or 8-HDF. This suggests that the binding pocket is not conserved. Our study proposes that ErCRY4a only harbor one light-sensitive cofactor, which in turn suggests a functional specialization different from most photolyases.

The human ATP-binding cassette (ABC) transporter, ABCG2 is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Forster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps. HighlightsO_LIABCG2 is a multidrug pump which moves between states having low or high affinity for substrates and inhibitors C_LIO_LIWe introduce a time-resolved Forster resonance energy transfer assay to measure interaction of substrates and inhibitors to ABCG2 C_LIO_LIWe confirm that NBD dimerization is associated with a switch from a high to a low affinity site for an ABCG2 inhibitor C_LI

The p75 neurotrophin receptor (p75NTR) is an important mediator of synaptic depression and neuronal cell death, and its expression increases upon nerve injury and in neurodegenerative diseases. However, the molecular mechanisms leading to the activation of this receptor are still a matter of debate. The oligomerization properties of the death domain (DD) of p75NTR are critical for our understanding of the activation mechanisms of the receptor. In this paper, we present additional evidence supporting the existence of an equilibrium between monomeric and dimeric forms of the p75NTR DD in solution and in the absence of any other protein. Dynamic light scattering (DLS) measurements of native, untagged human p75NTR DD at room temperature yielded Rh=2.11 for this domain in 20mM phosphate buffer, corresponding to a molecular weight (MW) of approximately 19kDa, much closer to the theoretical MW of the homodimer (i.e. 21kDa) than the monomer. MWs deduced from the Rh of different control proteins used as standards were all congruent with their theoretical MWs. In addition, size-exclusion FPLC profiles of un-tagged human p75NTR DD in both HEPES and phosphate buffers revealed elution volumes corresponding to a MW of about 15kDa, which is intermediate between monomer and dimer, and indicative of dynamic monomer/dimer interconversion during the run. Together with our previous NMR studies, as well as biophysical data for other investigators, these results support the notion that the DD of p75NTR exists in equilibrium between monomers and dimers in solution, a notion that is in agreement with the oligomerization properties of all members of the DD superfamily.

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to combat cancer is increasingly being investigated using a variety of small molecule inhibitors. Clinical success for these inhibitors has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Although mitochondrial oxidative metabolism is essential for malignant growth, mitochondria OXPHOS is also essential to the physiology of all organs, including high-energy-demand organs like the heart. In comparing tumor OXPHOS reliance to these preeminent oxidative organs it is unclear if a therapeutic window for targeting mitochondrial OXPHOS in cancer exists. To address this gap in knowledge, mitochondrial OXPHOS was comprehensively evaluated across various murine tumors and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were consistently lower across distinct tumor types. Operating on the assumption that intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across tumor types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given tumor type.

Mixed-type enzyme inhibitors were originally envisaged to decrease the enzyme affinity for the substrates and the maximum turnover rate by simultaneously targeting two distinct protein sites, i.e., the active site and an allosteric site. After a century from the first formulation of this hypothesis, the consensus on its validity is still unanimous, although several of its implications are in open conflict with the current knowledge on molecular recognition mechanisms. In particular, there is no plausible explanation for the experimental evidence that mixed-type inhibitors bind the enzyme active sites always more effectively than the allotopic sites. In an attempt to solve this controversy, it was found that the preference of mixed inhibitors for active sites emerges as an inevitable numerical artifact that is implicit in the equations used to model the apparent mixed inhibition caused under certain circumstances by active site-bound competitive inhibitors. Hence, proving that the consolidated model of mixed inhibition is incorrect and, more generally, strongly pointing to the biological irrelevance of mixed-type inhibition.

Giardia intestinalis is a protozoan parasite causing giardiasis, a severe, sometimes even life-threatening, diarrheal disease. Giardia is one of only a few known organisms that lack de novo synthesis of DNA building blocks, and the parasite is therefore completely dependent on salvaging deoxyribonucleosides from the host. The deoxyribonucleoside kinases (dNKs) needed for this salvage are generally divided into two structurally distinct families, thymidine kinase 1 (TK1)-like dNKs and non-TK1-like dNKs. We have characterized the G. intestinalis deoxyadenosine kinase and found that it, in contrast to previously studied non-TK1-like dNKs, has a tetrameric structure. Deoxyadenosine was the best natural substrate of the enzyme (KM=1.12 M; Vmax=10.3 mol{middle dot}min-1{middle dot}mg-1), whereas the affinities for deoxyguanosine, deoxyinosine and deoxycytidine were 400-2000 times lower. Deoxyadenosine analogues halogenated at the 2- and/or 2 s-positions were also potent substrates, with comparable EC50 values as the main drug used today, metronidazole, but with the advantage of being usable on metronidazole-resistant parasites. Cryo-EM and 2.1 [A] X-ray structures of the enzyme in complex with the product dAMP (and dADP) showed that the tetramer is kept together by extended N- and C-termini that reach across from one canonical dimer to the next in a novel dimer-dimer interaction. Removal of the two termini resulted in lost ability to form tetramers and a 100-fold decreased deoxyribonucleoside substrate affinity. This is the first example of a non-TK1-like dNK that has a higher substrate affinity as the result of a higher oligomeric state. The development of high substrate affinity could be an evolutionary key factor behind the ability of the parasite to survive solely on deoxyribonucleoside salvage. Authors summaryThe human pathogen Giardia intestinalis is one of only a few organisms that lack ribonucleotide reductase and is therefore completely dependent on salvaging deoxyribonucleosides from the host for the supply of DNA building blocks. We have characterized one of the G. intestinalis salvage enzymes, which was named deoxyadenosine kinase based on its substrate specificity. The enzyme also phosphorylated many deoxyadenosine analogues that were equally efficient in preventing parasite growth as the most used drug today, metronidazole, and also usable against metronidazole-resistant parasites. Structural analysis of the enzyme with cryo-EM and X-ray crystallography showed that the enzyme was unique in its family of deoxyribonucleoside kinases by forming a tetramer and mutational analysis showed that tetramerization is a prerequisite for the high substrate affinity of the enzyme. The ability to gain substrate affinity by increasing the number of enzyme subunits could potentially represent an evolutionary pathway that has assisted the parasite to become able to survive entirely on salvage synthesis of DNA building blocks.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, both in sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with heme substrate, no structures have been reported of IsdG-type proteins in complex with product, unlike HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IX (BV) rather than the wild-type (WT) mycobilin isomers as product. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product BV as a proxy to study the IsdG-type protein/product complex. First we show that BV has nanomolar affinity for MhuD and the R26S variant. Second we determined the MhuD-R26S-BV complex structure to 2.5 [A], which reveals two notable features (1) two BV molecules bound per active site and (2) a new -helix (3) as compared with the MhuD-heme structure. Finally, by molecular dynamics simulations we show that 3 is stable with the proximal BV alone. MhuDs high affinity for its product and structural and electrostatic changes that accompany substrate turnover suggest that there is an unidentified protein that is responsible for product extraction from MhuD and other IsdG-type proteins.

S-acylation is a common post-translational modification of membrane protein cysteine residues with many regulatory roles. S-acylation adjacent to transmembrane domains has been described in the literature as affecting diverse protein properties including turnover, trafficking and microdomain partitioning. However, all of these data are derived from mammalian and yeast systems. Here we examine the role of S-acylation adjacent to the transmembrane domain of the plant pathogen perceiving receptor-like kinase FLS2. Surprisingly, S-acylation of FLS2 adjacent to the transmembrane domain is not required for either FLS2 trafficking or signalling function. Expanding this analysis to the wider plant receptor-like kinase superfamily we find that S-acylation adjacent to receptor-like kinase domains is common but poorly conserved between orthologues through evolution. This suggests that S-acylation of receptor-like kinases at this site is likely the result of chance mutation leading to cysteine occurrence. As transmembrane domains followed by cysteine residues are common motifs for S-acylation to occur, and many S-acyl transferases appear to have lax substrate specificity, we propose that many receptor-like kinases are fortuitously S-acylated once chance mutation has introduced a cysteine at this site. Interestingly some receptor-like kinases show conservation of S-acylation sites between orthologues suggesting that S-acylation has come to play a role and has been positively selected for during evolution. The most notable example of this is in the ERECTA-like family where S-acylation of ERECTA adjacent to the transmembrane domain occurs in all ERECTA orthologues but not in the parental ERECTA-like clade. This suggests that ERECTA S-acylation occurred when ERECTA emerged during the evolution of angiosperms and may have contributed to the neo-functionalisation of ERECTA from ERECTA-like proteins.

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and [~]3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

Here we report an adapted protocol using the Promega NAD/NADH-Glo Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3- acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate. In fact, AcPyrAD generates amplification signals of larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where AcPyrAD is the product. We also propose an application of this method based on competition between AcPyr and other free bases to rank their preference for SARM1. This has significant advantages over traditional methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base exchange activity.

Intracellular heme formation and trafficking are fundamental processes in living organisms. Three biogenesis pathways are used by bacteria and archaea to produce iron protoporphyrin IX (heme b) that diverge after the formation of the common intermediate uroporphyrinogen III (urogen III). In this work, we identify and provide a detailed characterization of the enzymes involved in the transformation of urogen III into heme. We show that in this organism operates the protoporphyrin-dependent pathway (PPD pathway), in which the last reaction is the incorporation of ferrous iron into the porphyrin ring by the ferrochelatase enzyme. In general, following this final reaction, little is known about how the formed heme b reaches the target proteins. In particular, the chaperons that are thought to be required to traffic heme for incorporation into hemeproteins to avoid the cytotoxicity associated to free heme, remain largely unidentified. We identified in C. jejuni a chaperon-like protein, named CgdH2, that binds heme with a dissociation constant of 4.9 {+/-} 1.0 {micro}M, a binding that is impaired upon mutation of residues histidine 45 and 133. We show that C. jejuni CgdH2 establishes protein-protein interactions with ferrochelatase, which should enable for the observed transfer of heme from ferrochelatase to CgdH2. Phylogenetic analysis revealed that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is a novel chaperone and the first protein identified as an acceptor of the intracellularly formed heme, thus enlarging our understanding of bacterial heme homeostasis.

The MYC associated factor X (MAX) is the heterodimeric partner of the MYC paralogs (MYC, MYCN and MYCL). When deregulated, high level of the MYC paralogs contribute to all aspects of tumorigenesis and tumor growth. MAX can also heterodimerize with the MXD proteins, MNT and MGA. Heterodimerization and sequence specific DNA binding to the E-Box sequences at gene promoters is controlled by their heterodimerization with the MAX b-HLH-LZ. As a heterodimer with MAX, MYC proteins activate genes involved in cell metabolism, growth and proliferation whereas MXD proteins, MNT and MGA repress them. MAX can also bind to the E-Bos sequence as a homodimer. Being devoid of a transactivation domain it can act as an antagonist of the MYC/MAX heterodimers. Variants of MAX have been reported to be linked to cancer. These variants are either not expressed, inactivated or lead to missense mutations. This has led to the notion that MAX may have a tumor suppressor role. Here, we characterize three of those variants with missense mutations in the basic region, i.e. E32K, R35P and R35C. We analyzed their heterodimerization with the b-HLH-LZ of MYC and their DNA binding properties as homo-and heterodimers. The R35C variant b-HLH-LZ was found to have a markedly increased affinity for the b-HLH-LZ of MYC. We also observed that all three b-HLH-LZ variants have a lower affinity as homodimers for the E-Box than the WT. This was shown to lead to a preferential binding of all the heterodimeric b-LHLH-LZ to the E-Box. This effect is exacerbated in the case of the R35C variant. We argue that this preferential binding of MYC as heterodimers with these variants to E-Box sequences could contribute to tumorigenesis. Hence, our results suggest that, mechanistically, the MAX homodimer bound to the E-Box could act as a tumor suppressor. MATERIALS AND METHODSO_ST_ABSMolecular modelingC_ST_ABSThe open source version 1.7.6.0 of Pymol was used for modeling and molecular rendering [1]. The crystal structure of the MAX homodimer bound to the E-Box (1HLO [2]) was used as a template for the generation of the models. The variants were generated using the mutagenesis function in the wizard. The conformation of the K32 side chain was manually set in order to avoid introducing steric clashes with DNA. Protein expression and purificationThe cDNA, coding for the MAX b-HLH-LZ (Max* hereafter, residues 22-103, UniProt entry P61244-1) to which are added the GSGC residues in c-terminal, inserted in the pET3a vector was already available in the laboratory [3] and was used as a template to generate the plasmids with inserts coding for each of the mutants (E32K, R35C and R35P) through quick-change PCR with Q5 DNA polymerase and DpnI from New England Biolabs. The primers used were purchased from IDT DNA, their sequences are listed in Table S1. Sequence for each construct was confirmed by Sanger sequencing at the Plateforme de sequencage SANGER - Centre de recherche du CHU de Quebec - Universite Laval. The primary structure for the basic region of each construct is given in Fig. 2A. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/715400v1_fig2.gif" ALT="Figure 2"> View larger version (41K): org.highwire.dtl.DTLVardef@1b05d5eorg.highwire.dtl.DTLVardef@1c1d692org.highwire.dtl.DTLVardef@ee469dorg.highwire.dtl.DTLVardef@15e0ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Structure schematics, specific and non-specific interactions dictating specificity and stability of binding of the basic region of MAX to the canonical (CACGTG) E-Box. A. Primary structure for the basic region of MAX and each of the variants. Positions making the most important contacts with the E-box are indicated by black arrows. Positions for the variants studied here are colored according to the Zappo colour scheme, following their physico-chemical properties: red for negative, blue for positive, magenta for proline and yellow for cysteine. B. The side chain (carboxylate) of E32 receives H-Bonds from the CA nucleobases in the leading strand (white carbon atoms). R35 and R36 make a salt bridges with phosphate groups while and the guanidino moiety of R36 makes a specific H-Bond with the nucleobase of the G in the strand of the reverse complement (cyan carbon atoms). C. The R35C mutation removes one non-specific salt-bridge at the interface of the complex. D. The aliphatic portion of the K side chain in the E32K variant is unable to accept the H-Bonds from the CA nucleobases and leads to the stabilisation of the complex and the helical structure of the basic region. E. In addition to removing a salt-bride, the Pro residue in the R35P kinks the path of the basic region, prevents the establishment of the specific H-Bonds mandatory for recognition of the E-Box and leads to unfolding of the helical state. C_FIG The MYC b-HLH-LZ (Myc*), the Max*WT b-HLH-LZ and its variants were expressed and purified as previously described [3,4] After lyophilisation, the b-HLH-LZs were kept at -20{degrees}C and solubilised in Myc buffer (50 mM NaCl, 50 mM NaH2PO4 pH 5.5) for Myc* or PBS for Max* at a final concentration of 1 mM before use. Circular dichroismAll circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier-type thermostat. The instrument was routinely calibrated using an aqueous solution of d-10-(+)-camphorsulfonic acid at 290.5 nm. Samples were prepared as follows: Max* (either WT or a variant) was diluted in 100 {micro}l 2X CD buffer (40 mM KCl, 11.4 mM K2HPO4, 28.6 mM KH2PO4, pH 6.8) and the volume adjusted to 106 {micro}l with PBS. 10 {micro}l TCEP 16 mM were added, and the volume further adjusted to 192 {micro}l with ddH2O before samples were incubated overnight at room temperature. After reduction, Myc* was added and the volume adjusted to 198 {micro}l with Myc buffer (Na2HPO4 0.95 mM, NaH2PO4 49.05 mM, 50 mM NaCl, pH 5.5). The DNA complexes were prepared as follows. After a 10 minutes incubation of the protein samples at room temperature, 0, 1 or 2 {micro}l of 2 mM of specific or non-specific DNA duplexes in 10 mM Tris pH 8.0 were added and the volume adjusted to 200 {micro}l with 10 mM Tris pH 8.0. The strands of the specific probe were: 5-ATT ACC CAC GTG TCC T*AC-3 and 5-GTA GGA CAC GTG GGT* AAT-3 (with the E-box sequence underlined) and the non-specific probe: 5-ATT ACC TCC GGA TCC T*AC-3 and 5-GTA GGA TCC GGA GGT* AAT-3 (Integrated DNA Technologies). Samples were further incubated for 10 minutes at room temperature and transferred to a 1 mm path length quartz cuvette. All spectra were recorded from 250 to 195 nm at 0.1 nm intervals by accumulating 10 spectra at 25 {degrees}C. Thermal denaturations were recorded at 222 nm from 5 to 95 {degrees}C at a heating rate of 1 {degrees}C/min. CD signal for spectra and thermal denaturations was corrected by substracting the signal from corresponding spectra or thermal denaturation either for buffer alone or the appropriate DNA duplex. CD signal was then converted to mean residue ellipticity using the following formula [5]: [{theta}] = {delta} {middle dot} MRW/(10{middle dot}c l) where [{theta}] is the mean residue ellipticity in deg {middle dot} cm2 dmol-1, {delta} is the CD signal in millidegrees, MRW is the mean residue weight, c is the concentration in mg/ml and l is the pathlength in mm. For the heterodimers, the concentration used was the sum of Max* and Myc* and the MRW was determined using a weighted average.