Biochemical Journal

Thermolysin, a bacterial zinc metalloprotease, has been previously been reported to exhibit a biphasic kinetic temperature dependence of kcat with a characteristic convex shape. This convex shaping is observed for almost all enzymes which display an Arrhenius break; fumarase is the exception with concave shaping. Here, thermolysin kinetics measured with the tripeptide substrate N-[3-(2-furyl)acryloyl]-Phe-Leu-Ala (FAFLA) resulted in a concave Arrhenius plot, characterized by a 30 kJ/mol increase in enthalpy and entropy of activation, in contrast to the typical 30 kJ/mol decrease. Although the shape of the Arrhenius break differs, ionic strength and macromolecular crowding both attenuate the energetic magnitude of the break point, consistent with prior work. It was hypothesized that a different step of the catalytic cycle of thermolysin was represented by kcat with FAFLA to give rise to this new behavior. A 91% dependence of kcat on viscosity and modest solvent isotope effects, both distinct from previously-characterized substrates, indicated that a physical step was responsible for the observed Arrhenius concavity. Hinge bending conformational changes of thermolysin, monitored using the phosphoramidon inhibitor (a FAFLA mimic), exhibited a fully linear temperature dependence, excluding these large-scale motions as the origin of concavity. It was therefore proposed that release of the N-[3-(2-furyl)acryloyl]-Phe product is likely rate limiting since release was proposed to involve a two-step pathway to free the product coordinated to the catalytic Zn2+ of thermolysin. These findings provide a mechanistic framework for seldom-seen concave break point behavior and insights into the contribution of dynamics of physical processes to catalysis. IMPORTANCE AND IMPACTEnzymes which display Arrhenius break behavior provide insight into how dynamics impact catalysis. Almost every enzyme thus far displays convex biphasic shape, with concave shaping often not acknowledged. Thermolysin, which previously only showed convex shaping, displayed concave behavior with a tripeptide substrate. By linking this unusual kinetic behavior to a physical, not chemical, process, this work highlights the possible origin of a rare phenomenon which can expand understanding of protein dynamics and biphasic Arrhenius behavior.

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.

Artemisinin is an effective antimalarial drug sourced from Artemisia annua, but its low and variable yields require enhancement either semi-synthetically or in-planta to meet the global demand for treatment. Though essential enzymes have been identified in the artemisinin biosynthetic pathway, including an essential Cytochrome P450 monooxygenase (CYP71AV1), there are still many unknowns. Cytochrome P450 reductase 1 (herein, AaCPR1), has been experimentally confirmed as an electron transfer partner for CYP71AV1 in its three step oxygenation of key artemisinin precursors. However, the recent discovery of a highly related CPR, herein AaCPR2, introduces the possibility that another, potentially more catalytically favourable interaction, could exist for CYP71AV1. Therefore, enzyme kinetics and differential scanning fluorimetry (DSF) were used in the characterisation of both AaCPR1 and AaCPR2 to determine the existence and source of their catalytic differences. Tested enzyme activity under cytochrome c and NADPH concentrations revealed that AaCPR1 had lower Km and higher kcat/Km values, while AaCPR2 had higher Vmax and kcat values. This suggests that AaCPR1 is more effective at reducing cytochrome c when substrate conditions are limiting, whereas AaCPR2 is more effective than AaCPR1 at reducing cytochrome c when substrate conditions are saturating. This implies a functional partitioning of the two enzymes on the basis of substrate availability. The DSF results provided deeper insight into the different protein-ligand interactions between the two enzymes. AaCPR2 reached lower maximum melting temperatures across all tested conditions, whereas AaCPR1 had higher maximum melting temperatures. Thus, AaCPR1 exhibits higher thermal stability and has a higher temperature threshold than AaCPR2. This contributes to the notion that the AaCPRs are functionally divergent also on the basis of temperature. The cumulative differences in melting behaviour between the two enzymes led to the hypothesis that AaCPR1 and AaCPR2 exhibit different domain motions that may lead to preferential catalysis for one redox partner over another. This was further supported by the prediction of a highly variable loop region between the two enzymes at the connecting domain just after the flexible hinge. If such loops are highly mobile, as predicted, then the residue differences therein could provide a bio-structural basis for the kinetic and thermal/biophysical differences observed between AaCPR1 and AaCPR2. These data support that AaCPR1 and AaCPR2 possess fundamental biophysical differences despite their high degree of relatedness. Ultimately, these differences suggest differential metabolic functions of the two enzyme in artemisinin biosynthesis and/or other important secondary metabolic processes.

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.

S-acylation is the addition of fatty acids to cysteine residues to regulate protein function and localization. S-acylation is catalyzed by the ZDHHC (Asp-His-His-Cys) family of protein S-acyltransferases (PATs), which S-acylate protein substrates by first auto-S-acylating the catalytic cysteine of the DHHC active site followed by transfer to the substrate. ZDHHC13 and ZDHHC17 are related ankyrin repeat domain (ANK) PATs that S-acylate multiple neuronal proteins, including huntingtin (HTT), the protein mutated in Huntington disease. However, unlike ZDHHC17 and other human PATs, ZDHHC13 possesses a non-canonical DQHC active site. As the first histidine is essential for auto-S-acylation, it is unclear if ZDHHC13 is catalytically active. Our phylogenetic analysis of eukaryotic ANK-containing PATs shows that ZDHHC13 orthologues are more divergent compared to ZDHHC17. While the ZDHHC17 DHHC is highly conserved, the motif varies among ZDHHC13 orthologues, with some vertebrate lineages containing a serine in place of the catalytic cysteine. Interestingly, we found that the ZDHHC13 S-acylation is lower than that of ZDHHC17, but the ZDHHC13 catalytic cysteine is indeed S-acylated. While expression of wild type (WT) ZDHHC13 in ZDHHC13 deficient HEK293T cells increased S-acylation of a HTT1-588 fragment, surprisingly, expression of catalytically dead DQHS ZDHHC13 was still able to facilitate HTT1-588 S-acylation equally. This suggests the ZDHHC13 catalytic cysteine is not required for S-acylation of target proteins, suggesting ZDHHC13 may coordinate another PAT. Indeed, we identified ZDHHC13 in high-molecular weight complexes. Our results indicate that ZDHHC13 is a likely pseudoenzyme that may function via a non-conventional mechanism reliant on other PATs. This work broadens our understanding of the function of this non-canonical PAT.