Computational remodeling of an enzyme conformational landscape for altered substrate selectivity.

Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design of hinge-mediated domain motions. Using this method, we redesign the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate, resulting in altered substrate selectivity. Steady-state kinetics of designed variants reveals activity increases with the non-native substrate of approximately 100-fold and selectivity switches of up to 1900-fold. Structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirm that conformational equilibria favor the target conformation. Our computational approach opens the door to targeted alterations of conformational states and equilibria, which should facilitate the design of biocatalysts with customized activity and selectivity.

TReSR: A PCR-compatible DNA sequence design method for engineering proteins containing tandem repeats.

Protein tandem repeats (TRs) are motifs comprised of near-identical contiguous sequence duplications. They are found in approximately 14% of all proteins and are implicated in diverse biological functions facilitating both structured and disordered protein-protein and protein-DNA interactions. These functionalities make protein TR domains an attractive component for the modular design of protein constructs. However, the repetitive nature of DNA sequences encoding TR motifs complicates their synthesis and mutagenesis by traditional molecular biology workflows commonly employed by protein engineers and synthetic biologists. To address this challenge, we developed a computational protocol to significantly reduce the complementarity of DNA sequences encoding TRs called TReSR (for Tandem Repeat DNA Sequence Redesign). The utility of TReSR was demonstrated by constructing a novel constitutive repressor synthesized by duplicating the LacI DNA binding domain into a single-chain TR construct by assembly PCR. Repressor function was evaluated by expression of a fluorescent reporter delivered on a single plasmid encoding a three-component genetic circuit. The successful application of TReSR to construct a novel TR-containing repressor with a DNA sequence that is amenable to PCR-based construction and manipulation will enable the incorporation of a wide range of TR-containing proteins for protein engineering and synthetic biology applications.

A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30-40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9's prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish PCSK9's ability to bind LDL reported here supports the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.

Origin of conformational dynamics in a globular protein.

Protein structures are dynamic, undergoing motions that can play a vital role in function. However, the link between primary sequence and conformational dynamics remains poorly understood. Here, we studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual core-residue substitutions in DANCER-3, a streptococcal protein G domain ß1 variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Using a combination of solution NMR experiments and molecular dynamics simulations, we demonstrate that only two mutations are necessary to create this conformational exchange, and that these mutations work synergistically, with one destabilizing the native structure and the other allowing two new conformational states to be accessed on the energy landscape. Overall, our results show how dynamics can appear in a stable globular fold, a critical step in the molecular evolution of dynamics-linked functions.

Antimicrobial peptide LL-37 and its truncated forms, GI-20 and GF-17, exert spermicidal effects and microbicidal activity against Neisseria gonorrhoeae.

STUDY QUESTION: Do the truncated LL-37 peptides, GI-20 and GF-17, have spermicidal activity and microbicidal effects on the sexually transmitted infection (STI) pathogen Neisseria gonorrhoeae with equivalent potency to LL-37? SUMMARY ANSWER: GI-20 and GF-17 exhibited spermicidal effects on both mouse and human sperm as well as microbicidal action on N. gonorrhoeae with the same efficacy as LL-37. WHAT IS KNOWN ALREADY: The antimicrobial peptide LL-37 exerts microbicidal activity against various STI pathogens as well as spermicidal effects on both mouse and human sperm. STUDY DESIGN, SIZE, DURATION: Spermicidal activities of GI-20 and GF-17 were evaluated in vitro in mouse and human sperm and in vivo in mice. Finally, in vitro antimicrobial effects of LL-37, GI-20 and GF-17 on an STI pathogen, N. gonorrhoeae were determined. All experiments were repeated three times or more. In particular, sperm samples from different males were used on each experimental day. PARTICIPANTS/MATERIALS, SETTING, METHODS: The plasma membrane integrity of peptide-treated sperm was assessed by cellular exclusion of Sytox Green, a membrane impermeable fluorescent DNA dye. Successful mouse in vitro fertilization was revealed by the presence of two pronuclei in oocytes following co-incubation with capacitated untreated/peptide-pretreated sperm. Sperm plus each peptide were transcervically injected into female mice and the success of in vivo fertilization was scored by the formation of 2-4 cell embryos 42 h afterward. Reproductive tract tissues of peptide pre-exposed females were then assessed histologically for any damage. Minimal inhibitory/bactericidal concentrations of LL-37, GI-20 and GF-17 on N. gonorrhoeae were determined by a standard method. MAIN RESULTS AND THE ROLE OF CHANCE: Like LL-37, treatment of sperm with GI-20 and GF-17 resulted in dose-dependent increases in sperm plasma membrane permeabilization, reaching the maximum at 18 and 3.6 µM for human and mouse sperm, respectively (P < 0.0001, as compared with untreated sperm). Mouse sperm treated with 3.6 µM GI-20 or GF-17 did not fertilize oocytes either in vitro or in vivo. Moreover, reproductive tract tissues of female mice pre-exposed to 3.6 µM GI-20 or GF-17 remained intact with no lesions, erosions or ulcerations. At 1.8-7.2 µM, LL-37, GI-20 and GF-17 exerted bactericidal effects on N. gonorrhoeae. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration of the inhibitory effects of GI-20 and GF-17 on human in vitro and in vivo fertilization cannot be performed due to ethical issues. WIDER IMPLICATIONS OF THE FINDINGS: Like LL-37, GI-20 and GF-17 acted as spermicides and microbicides against N. gonorrhoeae, without adverse effects on female reproductive tissues. With lower synthesis costs, GI-20 and GF-17 are attractive peptides for further development into vaginal spermicides/microbicides. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Canadian Institutes of Health Research (MOP119438 and CCI82413 to N.T.) and NIH (R01 AI105147 to G.W.). There are no competing interests to declare.

Rational design of proteins that exchange on functional timescales.

Proteins are intrinsically dynamic molecules that can exchange between multiple conformational states, enabling them to carry out complex molecular processes with extreme precision and efficiency. Attempts to design novel proteins with tailored functions have mostly failed to yield efficiencies matching those found in nature because standard methods do not allow the design of exchange between necessary conformational states on a functionally relevant timescale. Here we developed a broadly applicable computational method to engineer protein dynamics that we term meta-multistate design. We used this methodology to design spontaneous exchange between two novel conformations introduced into the global fold of Streptococcal protein G domain ß1. The designed proteins, named DANCERs, for dynamic and native conformational exchangers, are stably folded and switch between predicted conformational states on the millisecond timescale. The successful introduction of defined dynamics on functional timescales opens the door to new applications requiring a protein to spontaneously access multiple conformational states.

Dissecting the role of conformational change and membrane binding by the bacterial cell division regulator MinE in the stimulation of MinD ATPase activity.

The bacterial cell division regulators MinD and MinE together with the division inhibitor MinC localize to the membrane in concentrated zones undergoing coordinated pole-to-pole oscillation to help ensure that the cytokinetic division septum forms only at the mid-cell position. This dynamic localization is driven by MinD-catalyzed ATP hydrolysis, stimulated by interactions with MinE's anti-MinCD domain. This domain is buried in the 6-ß-stranded MinE "closed" structure, but is liberated for interactions with MinD, giving rise to a 4-ß-stranded "open" structure through an unknown mechanism. Here we show that MinE-membrane interactions induce a structural change into a state resembling the open conformation. However, MinE mutants lacking the MinE membrane-targeting sequence stimulated higher ATP hydrolysis rates than the full-length protein, indicating that binding to MinD is sufficient to trigger this conformational transition in MinE. In contrast, conformational change between the open and closed states did not affect stimulation of ATP hydrolysis rates in the absence of membrane binding, although the MinD-binding residue Ile-25 is critical for this conformational transition. We therefore propose an updated model where MinE is brought to the membrane through interactions with MinD. After stimulation of ATP hydrolysis, MinE remains bound to the membrane in a state that does not catalyze additional rounds of ATP hydrolysis. Although the molecular basis for this inhibited state is unknown, previous observations of higher-order MinE self-association may explain this inhibition. Overall, our findings have general implications for Min protein oscillation cycles, including those that regulate cell division in bacterial pathogens.

Impact of Differential Detergent Interactions on Transmembrane Helix Dimerization Affinities.

Interactions between transmembrane (TM) helices play a critical role in the fundamental processes required for cells to communicate and exchange materials with their surroundings. Our understanding of the factors that promote TM helix interactions has greatly benefited from our ability to study these interactions in the solution phase through the use of membrane-mimetic micelles. However, less is known about the potential influence of juxtamembrane regions flanking the interacting TM helices that may modulate dimerization affinities, even when the interacting surface itself is not altered. To investigate this question, we used solution NMR to quantitate the dimerization affinity of the major coat protein from the M13 bacteriophage in sodium dodecyl sulfate (SDS), a well-characterized model of a single-spanning self-associating TM protein. Here, we showed that a shorter construct lacking the N-terminal amphipathic helix has a higher dimerization affinity relative to that of the full-length protein, with no change in the helical structure between the monomeric and dimeric states in both cases. Although this translated into a 0.6 kcal/mol difference in free energy when the SDS solvent was approximated as a continuous phase, there were deviations from this model at high protein to detergent ratios. Instead, the equilibria were better fit to a model that treats the empty micelle as an active participant in the reaction, giving rise to standard free energies of association that were the same for both full-length and TM-segment constructs. According to this model, the higher apparent affinity of the shorter peptide could be completely explained by the enhanced detergent binding by the monomer relative to that bound by the dimer. Therefore, differential detergent binding between the monomeric and dimeric states provides a mechanism by which TM helix interactions can be modulated by noninteracting juxtamembrane regions.

Prediction of Stable Globular Proteins Using Negative Design with Non-native Backbone Ensembles.

Accurate predictions of protein stability have great potential to accelerate progress in computational protein design, yet the correlation of predicted and experimentally determined stabilities remains a significant challenge. To address this problem, we have developed a computational framework based on negative multistate design in which sequence energy is evaluated in the context of both native and non-native backbone ensembles. This framework was validated experimentally with the design of ten variants of streptococcal protein G domain ß1 that retained the wild-type fold, and showed a very strong correlation between predicted and experimental stabilities (R(2) = 0.86). When applied to four different proteins spanning a range of fold types, similarly strong correlations were also obtained. Overall, the enhanced prediction accuracies afforded by this method pave the way for new strategies to facilitate the generation of proteins with novel functions by computational protein design.

Activity-based profiling of the proteasome pathway during hepatitis C virus infection.

Hepatitis C virus (HCV) infection often leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The stability of the HCV proteins is controlled by ubiquitin-dependent and ubiquitin-independent proteasome pathways. Many viruses modulate proteasome function for their propagation. To examine the interrelationship between HCV and the proteasome pathways we employed a quantitative activity-based protein profiling method. Using this approach we were able to quantify the changes in the activity of several proteasome subunits and found that proteasome activity is drastically reduced by HCV replication. The results imply a link between the direct downregulation of the activity of this pathway and chronic HCV infection.

Influence of hydrophobic mismatch on the catalytic activity of Escherichia coli GlpG rhomboid protease.

Rhomboids comprise a broad family of intramembrane serine proteases that are found in a wide range of organisms and participate in a diverse array of biological processes. High-resolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid have provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. Key to this are observations that GlpG hydrophobic domain dimensions may not be sufficient to completely span the native lipid bilayer. This formed the basis for a model where hydrophobic mismatch Induces thinning of the local membrane environment to promote access to transmembrane substrates. However, hydrophobic mismatch also has the potential to alter the functional properties of the rhomboid, a possibility we explore in the current work. For this purpose, we purified the catalytic transmembrane domain of GlpG into phosphocholine or maltoside detergent micelles of varying alkyl chain lengths, and assessed proteolytic function with a model water-soluble substrate. Catalytic turnover numbers were found to depend on detergent alkyl chain length, with saturated chains containing 10-12 carbon atoms supporting maximal activity. Similar results were obtained in phospholipid bicelles, with no proteolytic activity being detected in longer-chain lipids. Although differences in thermal stability and GlpG oligomerization could not explain these activity differences, circular dichroism spectra suggest that mismatch gives rise to a small change in structure. Overall, these results demonstrate that hydrophobic mismatch can exert an inhibitory effect on rhomboid activity, with the potential for changes in local membrane environment to regulate activity in vivo.

Profiling Kinase Activity during Hepatitis C Virus Replication Using a Wortmannin Probe.

To complete its life cycle, the hepatitis C virus (HCV) induces changes to numerous aspects of its host cell. As kinases act as regulators of many pathways utilized by HCV, they are likely enzyme targets for virally induced inhibition or activation. Herein, we used activity-based protein profiling (ABPP), which allows for the identification of active enzymes in complex protein samples and the quantification of their activity, to identify kinases that displayed differential activity in HCV-expressing cells. We utilized an ABPP probe, wortmannin-yne, based on the kinase inhibitor wortmannin, which contains a pendant alkyne group for bioconjugation using bioorthogonal chemistry. We observed changes in the activity of kinases involved in the mitogen-activated protein kinase pathway, apoptosis pathways, and cell cycle control. These results establish changes to the active kinome, as reported by wortmannin-yne, in the proteome of human hepatoma cells actively replicating HCV. The observed changes include kinase activity that affect viral entry, replication, assembly, and secretion, implying that HCV is regulating the pathways that it uses for its life cycle through modulation of the active kinome.

Micelle-catalyzed domain swapping in the GlpG rhomboid protease cytoplasmic domain.

Three-dimensional domain swapping is a mode of self-interaction that can give rise to altered functional states and has been identified as the trigger event in some protein deposition diseases, yet rates of interconversion between oligomeric states are usually slow, with the requirement for transient disruption of an extensive network of interactions giving rise to a large kinetic barrier. Here we demonstrate that the cytoplasmic domain of the Escherichia coli GlpG rhomboid protease undergoes slow dimerization via domain swapping and that micromolar concentrations of micelles can be used to enhance monomer-dimer exchange rates by more than 1000-fold. Detergents bearing a phosphocholine headgroup are shown to be true catalysts, with hexadecylphosphocholine reducing the 26 kcal/mol free energy barrier by >11 kcal/mol while preserving the 5 kcal/mol difference between monomer and dimer states. Catalysis involves the formation of a micelle-bound intermediate with a partially unfolded structure that is primed for domain swapping. Taken together, these results are the first to demonstrate true catalysis for domain swapping, by using micelles that work in a chaperonin-like fashion to unfold a kinetically trapped state and allow access to the domain-swapped form.

A new chemical probe for phosphatidylinositol kinase activity.

Phosphatidylinositol kinases (PIKs) are key enzymatic regulators of membrane phospholipids and membrane environments that control many aspects of cellular function, from signal transduction to secretion, through the Golgi apparatus. Here, we have developed a photoreactive "clickable" probe, PIK-BPyne, to report the activity of PIKs. We investigated the selectivity and efficiency of the probe to both inhibit and label PIKs, and we compared PIK-BPyne to a wortmannin activity-based probe also known to target PIKs. We found that PIK-BPyne can act as an effective in situ activity-based probe, and for the first time, report changes in PI4K-IIIß activity induced by the hepatitis C virus. These results establish the utility of PIK-BPyne for activity-based protein profiling studies of PIK function in native biological systems.

Activity-based protein profiling of the Escherichia coli GlpG rhomboid protein delineates the catalytic core.

Rhomboid proteins comprise the largest class of intramembrane protease known, being conserved from bacteria to humans. The functional status of these proteases is typically assessed through direct or indirect detection of peptide cleavage products. Although these assays can report on the ability of a rhomboid to catalyze peptide bond cleavage, differences in measured hydrolysis rates can reflect changes in the structure and activity of catalytic residues, as well as the ability of the substrate to access the active site. Here we show that a highly reactive and sterically unencumbered fluorophosphonate activity-based protein profiling probe can be used to report on the catalytic integrity of active site residues in the Escherichia coli GlpG protein. We used results obtained with this probe on GlpG in proteomic samples, in combination with a conventional assay of proteolytic function on purified samples, to identify residues that are located on the cytoplasmic side of the lipid bilayer that are required for maximal proteolytic activity. Regions tested include the 90-residue aqueous-exposed N-terminus that encompasses a globular structure that we have determined by solution nuclear magnetic resonance, along with residues on the cytoplasmic side of the transmembrane domain core. While in most cases mutation or elimination of these residues did not significantly alter the catalytic status of the GlpG active site, the lipid-facing residue Arg227 was found to be important for maintaining a catalytically competent active site. In addition, we found a functionally critical region outside the transmembrane domain (TMD) core that is required for maximal protease activity. This region encompasses an additional 8-10 residues on the N-terminal side of the TMD core that precedes the first transmembrane segment and was not previously known to play a role in rhomboid function. These findings highlight the utility of the activity-based protein profiling approach for the characterization of rhomboid function.

Contemporary methods in structure determination of membrane proteins by solution NMR.

Integral membrane proteins are vital to life, being responsible for information and material exchange between a cell and its environment. Although high-resolution structural information is needed to understand how these functions are achieved, membrane proteins remain an under-represented subset of the protein structure databank. Solution NMR is increasingly demonstrating its ability to help address this knowledge shortfall, with the development of a diverse array of techniques to counter the challenges presented by membrane proteins. Here we document the advances that are helping to define solution NMR as an effective tool for membrane protein structure determination. Developments introduced over the last decade in the production of isotope-labeled samples, reconstitution of these samples into the growing selection of NMR-compatible membrane-mimetic systems, and the approaches used for the acquisition and application of structural restraints from these complexes are reviewed.

Regulation of symmetric bacterial cell division by MinE: What is the role of conformational dynamics?

Symmetric cell division in Gram-negative bacteria requires the concerted action of three Min proteins that together ensure exclusive formation of the cell division septum at the mid-point of the cell. We have recently described the structure and dynamic properties of MinE, the protein responsible for directing the cell division inhibitor complex formed by the MinC and MinD proteins away from the middle of the cell. An unexpected feature of this structure was the location of MinD-binding residues at buried, non-accessible sites in the dimeric interface. Here we elaborate on the potential role of conformational changes that might be involved to allow access to these residues, along with the interesting questions raised by these features of the MinE structure.

Appropriation of the MinD protein-interaction motif by the dimeric interface of the bacterial cell division regulator MinE.

MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cytokinetic septum to the midpoint of the cell in gram negative bacteria. Critical for this oscillation is MinD-binding by MinE to stimulate MinD ATP hydrolysis, a function that had been assigned to the first ∼30 residues in MinE. Previous models based on the structure of an autonomously folded dimeric C-terminal fragment suggested that the N-terminal domain is freely accessible for interactions with MinD. We report here the solution NMR structure of the full-length MinE dimer from Neisseria gonorrhoeae, with two parts of the N-terminal domain forming an integral part of the dimerization interface. Unexpectedly, solvent accessibility is highly restricted for residues that were previously hypothesized to directly interact with MinD. To delineate the true MinD-binding region, in vitro assays for MinE-stimulated MinD activity were performed. The relative MinD-binding affinities obtained for full-length and N-terminal peptides from MinE demonstrated that residues that are buried in the dimeric interface nonetheless participate in direct interactions with MinD. According to results from NMR spin relaxation experiments, access to these buried residues may be facilitated by the presence of conformational exchange. We suggest that this concealment of MinD-binding residues by the MinE dimeric interface provides a mechanism for prevention of nonspecific interactions, particularly with the lipid membrane, to allow the free diffusion of MinE that is critical for Min protein oscillation.

1H, 13C, 15N chemical shift assignments for the Neisseria gonorrhoeae MinE regulator of cell division septum placement.

MinE acts together with MinC and MinD to prevent placement of the cell division septum in the polar regions of gram negative bacteria, thereby ensuring that productive cell division occurs solely at the mid-cell. Here we report the backbone and side chain (1)H, (13)C and (15)N resonance assignments for MinE from Neisseria gonorrhoeae.