Substrate-induced Changes in Protease Active Site Conformation Impact on Subsequent Reactions with Substrates

Enzymatic catalysis of biochemical reactions is essential to all living systems. The “lock and key” and “induced fit” models were early contributions to our understanding of the mechanisms involved in the reaction between an enzyme and its substrate. However, whether a given substrate-induced conformation is rigid or remains flexible has not yet been determined. By measuring the enzyme activity and intrinsic fluorescence of a nonspecific Eisenia fetida protease-I with different chromogenic substrates, we show that in subsequent reactions of protease with substrates, both the “lock and key” and “induced fit” mechanisms are used depending on the degree of conformational change required. Chromozym-Th- or chromosym-Ch-induced protease conformations were unable to bind chromozym-U. The chromosym-U-induced protease conformation remained flexible and could be further induced by chromozym-Th and chromozym-Ch. When low concentrations of guanidine HCl were used to disturb the conformation of the enzyme, only small changes in intrinsic fluorescence of the chromozym-Th-induced protease were detected, in contrast to the native enzyme whose intrinsic fluorescence markedly increased. This indicates that the substrate-induced enzyme was relatively rigid compared with the native protease. Utilizing a lock and key mechanism for secondary substrate reactions may have adaptive value in that it facilitates high efficiency in enzymatic reactions.     

Conformational Plasticity of the Essential Membrane-associated Mannosyltransferase PimA from Mycobacteria

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the “open” and “closed” conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal “Rossmann fold” domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.     

The Structure of an NDR/LATS Kinase–Mob Complex Reveals a Novel Kinase–Coactivator System and Substrate Docking Mechanism

Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. “Hippo” pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with “Mob” coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1–Mob2, to our knowledge the first of an NDR/LATS kinase–Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1’s regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides.     

Variation in parasite resistance of Arctic charr,Salvelinus alpinus,between and within sympatric morphs

Genetic variation in resistance against parasite infections is a predominant feature in host–parasite systems. However, mechanisms maintaining genetic polymorphism in resistance in natural host populations are generally poorly known. We explored whether differences in natural infection pressure between resource‐based morphs of Arctic charr (Salvelinus alpinus) have resulted in differentiation in resistance profiles. We experimentally exposed offspring of two morphs from Lake Þingvallavatn (Iceland), the pelagic planktivorous charr (“murta”) and the large benthivorous charr (“kuðungableikja”), to their common parasite, eye fluke Diplostomum baeri, infecting the eye humor. We found that there were no differences in resistance between the morphs, but clear differences among families within each morph. Moreover, we found suggestive evidence of resistance of offspring within families being positively correlated with the parasite load of the father, but not with that of the mother. Our results suggest that the inherited basis of parasite resistance in this system is likely to be related to variation among host individuals within each morph rather than ecological factors driving divergent resistance profiles at morph level. Overall, this may have implications for evolution of resistance through processes such as sexual selection.     

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K_d∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of K_d values with K_m values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.     

Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition

N⁶-Methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism. Recently, human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Here, we report five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. Compared with other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded β-helix fold typical of this protein family. Among the unique features, a distinct “lid” region of Alkbh5 plays a vital role in substrate recognition and catalysis. An unexpected disulfide bond between Cys-230 and Cys-267 is crucial for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a “flipping” motif toward the central β-helix fold. We generated a substrate binding model of Alkbh5 based on a demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of α-ketoglutarate revealed that the active site cavity of Alkbh5 is much smaller than that of FTO and preferentially binds small molecule inhibitors. Taken together, our findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin

Human α₂-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M’s unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible “bait region.” As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the “tabula rasa” bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.     

Are British urban foxes (Vulpes vulpes) “bold”? The importance of understanding human–wildlife interactions in urban areas

Human–wildlife interactions are believed to be increasing in urban areas. In Britain, numerous media reports have stated that urban foxes (Vulpes vulpes) are becoming “bolder,” thereby posing a risk to public safety. However, such claims overlook how an individual''s personality might influence urban fox behavior. Personality determines multiple aspects of an animal''s interactions with both conspecifics and its environment, and can have a significant impact on how people perceive wildlife. Furthermore, describing urban foxes as “bold” confounds two different but inter‐related behaviors, both of which influence an animal''s propensity to take risks. Neophobia affects an animal''s reaction to novelty, wariness its reaction to potential threats. Since urban wildlife frequently encounters both novel and threatening stimuli, a highly adaptable species such as the red fox might be predicted to exhibit reduced neophobia and wariness. We investigated how social status influenced both behaviors in Bristol''s fox population. Dominant foxes were significantly more neophobic and warier than subordinates, which adopt a more exploratory and risk‐taking lifestyle to meet their energetic and other needs. We found no seasonal effect on neophobia and wariness, although this may be due to sample size. The presence of conspecifics decreased neophobia for dominants, and wariness for both dominants and subordinates. We highlight the importance of considering animal social status and personality when planning management protocols, since interventions that destabilize fox social groups are likely to increase the number of subordinate foxes in the population, thereby increasing rather than decreasing the number of interactions between humans and urban foxes.     

Acidification Asymmetrically Affects Voltage-dependent Anion Channel Implicating the Involvement of Salt Bridges

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to “gate” between an open and several “closed” states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pK_a values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.     

A non‐helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na⁺‐dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate‐bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly‐Met‐Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.     

Accurate homologous recombination is a prominent double-strand break repair pathway in mammalian chromosomes and is modulated by mismatch repair protein Msh2

We designed DNA substrates to study intrachromosomal recombination in mammalian chromosomes. Each substrate contains a thymidine kinase (tk) gene fused to a neomycin resistance (neo) gene. The fusion gene is disrupted by an oligonucleotide containing the 18-bp recognition site for endonuclease I-SceI. Substrates also contain a “donor” tk sequence that displays 1% or 19% sequence divergence relative to the tk portion of the fusion gene. Each donor serves as a potential recombination partner for the fusion gene. After stably transfecting substrates into mammalian cell lines, we investigated spontaneous recombination and double-strand break (DSB)-induced recombination following I-SceI expression. No recombination events between sequences with 19% divergence were recovered. Strikingly, even though no selection for accurate repair was imposed, accurate conservative homologous recombination was the predominant DSB repair event recovered from rodent and human cell lines transfected with the substrate containing sequences displaying 1% divergence. Our work is the first unequivocal demonstration that homologous recombination can serve as a major DSB repair pathway in mammalian chromosomes. We also found that Msh2 can modulate homologous recombination in that Msh2 deficiency promoted discontinuity and increased length of gene conversion tracts and brought about a severalfold increase in the overall frequency of DSB-induced recombination.     

DNA Damage Regulates Translation through β-TRCP Targeting of CReP

The Skp1-Cul1-F box complex (SCF) associates with any one of a number of F box proteins, which serve as substrate binding adaptors. The human F box protein βTRCP directs the conjugation of ubiquitin to a variety of substrate proteins, leading to the destruction of the substrate by the proteasome. To identify βTRCP substrates, we employed a recently-developed technique, called Ligase Trapping, wherein a ubiquitin ligase is fused to a ubiquitin-binding domain to “trap” ubiquitinated substrates. 88% of the candidate substrates that we examined were bona fide substrates, comprising twelve previously validated substrates, eleven new substrates and three false positives. One βTRCP substrate, CReP, is a Protein Phosphatase 1 (PP1) specificity subunit that targets the translation initiation factor eIF2α to promote the removal of a stress-induced inhibitory phosphorylation and increase cap-dependent translation. We found that CReP is targeted by βTRCP for degradation upon DNA damage. Using a stable CReP allele, we show that depletion of CReP is required for the full induction of eIF2α phosphorylation upon DNA damage, and contributes to keeping the levels of translation low as cells recover from DNA damage.     

Characterization of Tiki,a New Family of Wnt-specific Metalloproteases

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn²⁺/Co²⁺ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.     

The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding Functions

Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing,” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.     

Substrate Compliance versus Ligand Density in Cell on Gel Responses

Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on “rigid” glass or “stiff” gels than on “soft” gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.