Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease.

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from -31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.

Reframing prosegment-dependent folding and limits on natural protein folding landscapes from an evolutionary perspective.

In this perspective, we propose that the folding energy landscapes of model proteases including pepsin and alpha-lytic protease (αLP), which lack thermodynamic stability and fold on the order of months to millennia, respectively, should be viewed as not evolved and fundamentally distinct from their extended zymogen forms. These proteases have evolved to fold with prosegment domains and robustly self-assemble as expected. In this manner, general protein folding principles are strengthened. In support of our view, αLP and pepsin exhibit hallmarks of frustration associated with unevolved folding landscapes, such as non-cooperativity, memory effects, and substantial kinetic trapping. The evolutionary implications of this folding strategy are considered in detail. Direct applications of this folding strategy on enzyme design, finding new drug targets, and constructing tunable folding landscapes are also discussed. Together with certain proteases, growing examples of other folding "exceptions"-including protein fold switching, functional misfolding, and prevalent inability to refold-suggests a paradigm shift in which proteins may evolve to exist in a wide range of energy landscapes and structures traditionally thought to be avoided in nature.

Bioactivity of the cannabigerol cannabinoid and its analogues - the role of 3-dimensional conformation.

Cannabinoids are naturally occurring bioactive compounds with the potential to help treat chronic illnesses including epilepsy, Parkinson's disease, dementia and multiple sclerosis. Their general structures and efficient syntheses are well documented in the literature, yet their quantitative structure-activity relationships (QSARs), particularly 3-dimensional (3-D) conformation-specific bioactivities, are not fully resolved. Cannabigerol (CBG), an antibacterial precursor molecule for the most abundant phytocannabinoids, was characterised herein using density functional theory (DFT), together with selected analogues, to ascertain the influence of the 3D structure on their activity and stability. Results showed that the CBG family's geranyl chains tend to coil around the central phenol ring while its alkyl side-chains form H-bonds with the para-substituted hydroxyl groups as well as CH⋯π interactions with the aromatic density of the ring itself, among other interactions. Although weakly polar, these interactions are structurally and dynamically influential, effectively 'stapling' the ends of the chains to the central ring structure. Molecular docking of the differing 3-D poses of CBG to cytochrome P450 3A4 resulted in lowered inhibitory action by the coiled conformers, relative to their fully-extended counterparts, helping explain the trends in the inhibition of the metabolic activity of the CYP450 3A4. The approach detailed herein represents an effective method for the characterisation of other bioactive molecules, towards improved understanding of their QSARs and in guiding the rational design and synthesis of related compounds.

Precision cellular agriculture: The future role of recombinantly expressed protein as food.

Cellular agriculture is a rapidly emerging field, within which cultured meat has attracted the majority of media attention in recent years. An equally promising area of cellular agriculture, and one that has produced far more actual food ingredients that have been incorporated into commercially available products, is the use of cellular hosts to produce soluble proteins, herein referred to as precision cellular agriculture (PCAg). In PCAg, specific animal- or plant-sourced proteins are expressed recombinantly in unicellular hosts-the majority of which are yeast-and harvested for food use. The numerous advantages of PCAg over traditional agriculture, including a smaller carbon footprint and more consistent products, have led to extensive research on its utility. This review is the first to survey proteins currently being expressed using PCAg for food purposes. A growing number of viable expression hosts and recent advances for increased protein yields and process optimization have led to its application for producing milk, egg, and muscle proteins; plant hemoglobin; sweet-tasting plant proteins; and ice-binding proteins. Current knowledge gaps present research opportunities for optimizing expression hosts, tailoring posttranslational modifications, and expanding the scope of proteins produced. Considerations for the expansion of PCAg and its implications on food regulation, society, ethics, and the environment are also discussed. Considering the current trajectory of PCAg, food proteins from any biological source can likely be expressed recombinantly and used as purified food ingredients to create novel and tailored food products.

Aluminium catalysed oligomerisation in cement-forming silicate systems.

Alumino-silicates form the backbone of structural materials including cements and the concrete they form. However, the nanoscale aspects of the oligomerisation mechanisms elongating the (alumino-)silicate chains is not fully clarified; the role of aluminium in particular. Herein, we explore and contrast the growth of silicate and alumino-silicate oligomers by both neutral and anionic mechanisms, with focus on the influence of Al on oligomer structure and stability. Further, the spontaneity of chain lengthening in the absence and presence of Al of differing coordination (Al-IV, V, VI) was characterised. Result trends showed Al-IV facilitating oligomerisation in neutral conditions, with respect to Si only systems, effectively promoting longer chain formation and stabilisation. The anionic pathway similarly showed Al reducing the overall energetic barriers to oligomerisation. In both conditions, Al's coordinative and structural flexibility, at O-Al-O hinge points in particular, was responsible for the lowering of the energetic expense for oligomerisation. The results and implications resolved herein are informative for chain formation and stability for bulk material properties of alumino-silicate materials such as cements, where the aluminosilicate systems are dominated by short chains of 2-5 units in length.

Insights into the mechanism of membrane fusion induced by the plant defense element, plant-specific insert.

In plants, many natural defense mechanisms include cellular membrane fusion as a way to resist infection by external pathogens. Several plant proteins mediate membrane fusion, but the detailed mechanism by which they promote fusion is less clear. Understanding this process could provide valuable insights into these proteins' physiological functions and guide bioengineering applications (i.e. the design of antimicrobial proteins). The plant-specific insert (PSI) from Solanum tuberosum can help reduce certain pathogen attack via membrane fusion. To gain new insights into the process of PSI-induced membrane fusion, a combined approach of NMR, FRET, and in silico studies was used. Our results indicate that (i) under acidic conditions, the PSI experiences a monomer-dimer equilibrium, and the dimeric PSI induces membrane fusion below a certain critical pH; (ii) after fusion, the PSI resides in a highly dehydrated environment with limited solvent accessibility, suggesting its capability in reducing repulsive dehydration forces between liposomes to facilitate fusion; and (iii) as shown by molecular dynamics simulations, the PSI dimer can bind stably to membrane surfaces and can bridge liposomes in close proximity, a critical step for the membrane fusion. In summary, this study provides new and unique insights into the mechanisms by which the PSI and similar proteins induce membrane fusion.

Seed coat mucilages: Structural, functional/bioactive properties, and genetic information.

Seed coat mucilages are mainly polysaccharides covering the outer layer of the seeds to facilitate seed hydration and germination, thereby improving seedling emergence and reducing seedling mortality. Four types of polysaccharides are found in mucilages including xylan, pectin, glucomannan, and cellulose. Recently, mucilages from flaxseed, yellow mustard seed, chia seed, and so on, have been used extensively in the areas of food, pharmaceutical, and cosmetics contributing to stability, texture, and appearance. This review, for the first time, addresses the similarities and differences in physicochemical properties, molecular structure, and functional/bioactive properties of mucilages among different sources; highlights their structure and function relationships; and systematically summarizes the related genetic information, aiming with the intent to explore the potential functions thereby extending their future industrial applications.

Chlorogenic acid isomers directly interact with Keap 1-Nrf2 signaling in Caco-2 cells.

Chlorogenic acid (CGA) exists as multiple isomers (e.g., 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) in foods such as coffee beverages, fruits and vegetables. This study aimed to investigate relative activities of these six different CGA isomers to modify redox biology in inflamed Caco-2 cells that involved Nrf2 signaling. Caco-2 cells were pre-treated with individual CGA isomers to assess the relative effectiveness to mitigate oxidative stress. Isomer-specific capacity of different CGA isomers for direct free radical scavenging activity and potential endogenous control of oxidative stress were determined using chemical assays and cell-based experiments, respectively. Molecular dynamics simulations of the CGA and Keap1-Nrf2 complex were performed to predict CGA structure-specific interactions. Results demonstrated that dicaffeoylquinic acid (diCQA including 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) isomers had greater (p < 0.05) affinity to ameliorate oxidative stress through direct free radical scavenging activity. This observation corresponded to greater (p < 0.05) capacity to activate Nrf2 signaling compared to caffeoylquinic acid (CQA including 3-CQA, 4-CQA, and 5-CQA) isomers in inflamed differentiated Caco-2 cells. Simulations revealed that differences between the ability of CQA and diCQA to interact with the Keap1-Nrf2 complex may be due to differences in relative orientation within this complex. The observed CGA isomer-specific affinity for CQA to activate Nrf2 signaling was confirmed by nuclear translocation of Nrf2 induced by CGA and greater (p < 0.05) upregulation of genes related to Nrf2 expression.

The prosegment catalyzes native folding of Plasmodium falciparum plasmepsin II.

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77-124) and a longer (residues 65-124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding.

Comparative structure-function characterization of the saposin-like domains from potato, barley, cardoon and Arabidopsis aspartic proteases.

The present study characterized the aspartic protease saposin-like domains of four plant species, Solanum tuberosum (potato), Hordeum vulgare L. (barley), Cynara cardunculus L. (cardoon; artichoke thistle) and Arabidopsis thaliana, in terms of bilayer disruption and fusion, and structure pH-dependence. Comparison of the recombinant saposin-like domains revealed that each induced leakage of bilayer vesicles composed of a simple phospholipid mixture with relative rates Arabidopsis>barley>cardoon>potato. When compared for leakage of bilayer composed of a vacuole-like phospholipid mixture, leakage was approximately five times higher for potato saposin-like domain compared to the others. In terms of fusogenic activity, distinctions between particle size profiles were noted among the four proteins, particularly for potato saposin-like domain. Bilayer fusion assays in reducing conditions resulted in altered fusion profiles except in the case of cardoon saposin-like domain which was virtually unchanged. Secondary structure profiles were similar across all four proteins under different pH conditions, although cardoon saposin-like domain appeared to have higher overall helix structure. Furthermore, increases in Trp emission upon protein-bilayer interactions suggested that protein structure rearrangements equilibrated with half-times ranging from 52 to 120s, with cardoon saposin-like domain significantly slower than the other three species. Overall, the present findings serve as a foundation for future studies seeking to delineate protein structural features and motifs in protein-bilayer interactions based upon variability in plant aspartic protease saposin-like domain structures.

Foldase and inhibitor functionalities of the pepsinogen prosegment are encoded within discrete segments of the 44 residue domain.

Pepsin is initially produced as the zymogen pepsinogen, containing a 44 residue prosegment (PS) domain. When folded without the PS, pepsin forms a thermodynamically stable denatured state (refolded pepsin, Rp). To guide native folding, the PS binds to Rp, stabilizes the folding transition state, and binds tightly to native pepsin (Np), thereby driving the folding equilibrium to favor the native state. It is unknown whether these functionalities of the PS are encoded within the entire sequence or within discrete segments. PS residues 1p-29p correspond to a highly conserved region in pepsin-like aspartic proteases and we hypothesized that this segment is critical to PS-catalyzed folding. This notion was tested in the present study by characterizing the ability of various truncated PS peptides to bind Rp, catalyze folding from Rp to Np, and to inhibit Np. Four PS truncations were examined, corresponding to PS residues 1p-16p (PS1-16), 1p-29p (PS1-29), 17p-44p (PS17-44) and 30p-44p (PS30-44). The three PS functionalities could be ascribed primarily to discrete regions within the highly conserved motif: 1p-16p dictated Rp binding, 17p-29p dictated Np binding/inhibition, while the entire 1p-29p dictated transition state binding/catalyzing folding. Conversely, PS30-44 played no obvious role in PS-catalyzed folding; it is hypothesized that this more variable region may serve as a linker between PS1-29 and the mature domain. The high sequence conservation of PS1-29 and its role in catalyzing pepsin folding strongly suggest that there is a conserved PS-catalyzed folding mechanism shared by pepsin-like aspartic proteases with this motif.

Understanding the mechanism of prosegment-catalyzed folding by solution NMR spectroscopy.

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.

Engineered Nanoscale Food Ingredients: Evaluation of Current Knowledge on Material Characteristics Relevant to Uptake from the Gastrointestinal Tract.

The NanoRelease Food Additive project developed a catalog to identify potential engineered nanomaterials (ENMs) used as ingredients, using various food-related databases. To avoid ongoing debate on defining the term nanomaterial, NanoRelease did not use any specific definition other than the ingredient is not naturally part of the food chain, and its dimensions are measured in the nanoscale. Potential nanomaterials were categorized based on physical similarity; analysis indicated that the range of ENMs declared as being in the food chain was limited. Much of the catalog's information was obtained from product labeling, likely resulting in both underreporting (inconsistent or absent requirements for labeling) and/or overreporting (inability to validate entries, or the term nano was used, although no ENM material was present). Three categories of ingredients were identified: emulsions, dispersions, and their water-soluble powdered preparations (including lipid-based structures); solid encapsulates (solid structures containing an active material); and metallic or other inorganic particles. Although much is known regarding the physical/chemical properties for these ingredient categories, it is critical to understand whether these properties undergo changes following their interaction with food matrices during preparation and storage. It is also important to determine whether free ENMs are likely to be present within the gastrointestinal tract and whether uptake of ENMs may occur in their nanoform physical state. A practical decision-making scheme was developed to help manage testing requirements.

Inhibition of Plasmodium falciparum plasmepsins by drugs targeting HIV-1 protease: A way forward for antimalarial drug discovery.

Plasmodium species are causative agents of malaria, a disease that is a serious global health concern. FDA-approved HIV-1 protease inhibitors (HIV-1 PIs) have been reported to be effective in reducing the infection by Plasmodium parasites in the population co-infected with both HIV-1 and malaria. However, the mechanism of HIV-1 PIs in mitigating Plasmodium pathogenesis during malaria/HIV-1 co-infection is not fully understood. In this study we demonstrate that HIV-1 drugs ritonavir (RTV) and lopinavir (LPV) exhibit the highest inhibition activity against plasmepsin II (PMII) and plasmepsin X (PMX) of P. falciparum. Crystal structures of the complexes of PMII with both drugs have been determined. The inhibitors interact with PMII via multiple hydrogen bonding and hydrophobic interactions. The P4 moiety of RTV forms additional interactions compared to LPV and exhibits conformational flexibility in a large S4 pocket of PMII. Our study is also the first to report inhibition of P. falciparum PMX by RTV and the mode of binding of the drug to the PMX active site. Analysis of the crystal structures implies that PMs can accommodate bulkier groups of these inhibitors in their S4 binding pockets. Structurally similar active sites of different vacuolar and non-vacuolar PMs suggest the potential of HIV-1 PIs in targeting these enzymes with differential affinities. Our structural investigations and biochemical data emphasize PMs as crucial targets for repurposing HIV-1 PIs as antimalarial drugs.

The effect of thermal and ultrasonic treatment on amino acid composition, radical scavenging and reducing potential of hydrolysates obtained from simulated gastrointestinal digestion of cowpea proteins.

The effect of thermal and ultrasonic treatment of cowpea proteins (CP) on amino acid composition, radical scavenging and reducing potential of hydrolysates (CPH) obtained from in vitro simulated gastrointestinal digestion of CP was evaluated. Hydrolysis of native and treated CP with gastrointestinal pepsin and pancreatin yielded CPH that displayed antioxidant activities based on oxygen radical scavenging capacity (ORAC), ferric reducing antioxidant power (FRAP) and superoxide radical scavenging activity (SRSA). CPH derived from the treated CP yielded higher ORAC values than CPH from untreated proteins. However, lower significant FRAP and SRSA values were observed for these samples compared to untreated CPH (p < 0.05). Amino acid analysis indicated that CP processing decreased total sulphur-containing amino acids in the hydrolysates, particularly cysteine. The amount of cysteine appeared to be positively related to FRAP and SRSA values of CPH samples, but not ORAC. The results indicated that thermal and ultrasonic processing of CP can reduce the radical scavenging and reducing potential of the enzymatic hydrolysates possibly due to the decreased amounts of cysteine. Since the hydrolysates were generated with gastrointestinal enzymes, it is possible that the resulting compounds are produced to exert some health functions during normal consumption of cowpea.

Structure and mechanism of the saposin-like domain of a plant aspartic protease.

Many plant aspartic proteases contain an additional sequence of ~100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plant-specific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å, revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus.

Predicting global diet-disease relationships at the atomic level: a COVID-19 case study.

Over the past few months, numerous studies harnessed in silico methods such as molecular docking to evaluate food compounds for inhibitory activity against coronavirus infection and replication. These studies capitalize on the efficiency of computational methods to quickly guide subsequent research and examine diet-disease relationships, and their sudden widespread utility may signal new opportunities for future antiviral and bioactive food research. Using Coronavirus Disease 2019 (COVID-19) research as a case study, we herein provide an overview of findings from studies using molecular docking to study food compounds as potential inhibitors of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), explore considerations for the critical interpretation of study findings, and discuss how these studies help shape larger conversations of diet and disease.

Structures of plasmepsin X from Plasmodium falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme.

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

Dynamics of thermodynamically stable, kinetically trapped, and inhibitor-bound states of pepsin.

The pepsin folding mechanism involves a prosegment (PS) domain that catalyzes folding, which is then removed, resulting in a kinetically trapped native state. Although native pepsin (Np) is kinetically stable, it is irreversibly denatured due to a large folding barrier, and in the absence of the PS it folds to a more thermodynamically stable denatured state, termed refolded pepsin (Rp). This system serves as a model to understand the nature of kinetic barriers and folding transitions between compact states. Quasielastic neutron scattering (QENS) was used to characterize and compare the flexibility of Np, as a kinetically trapped state, with that of Rp, as a thermodynamically stable fold. Additionally, the dynamics of Np were compared with those of a partially unfolded form and a thermally stabilized, inhibitor-bound form. QENS revealed length-scale-dependent differences between Np and Rp on a picosecond timescale and indicated greater flexibility in Np, leading to the conclusion that kinetic stabilization likely does not correspond to reduced internal dynamics. Furthermore, large differences were observed upon inhibition, indicating that QENS of proteins in solution may prove useful for examining the role of conformational entropy changes in ligand binding.

Structural insights into the activation and inhibition of histo-aspartic protease from Plasmodium falciparum.

Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 Å resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.