Scott A. Hollingsworth

On the occurrence of linear groups in proteins

Linear groups—polypeptide conformations based on a single repeating ϕ,ψ‐pair—are a foundational concept in protein structure, yet how they are presented in textbooks is based largely on theoretical studies from the early days of protein structure analysis. Now, ultra‐high resolution protein structures provide a resource for an accurate empirical and systematic assessment of the linear groups that truly exist in proteins. Here, a purely conformation‐based survey of linear groups shows that only three distinct ϕ,ψ‐regions occur: a diverse set of extended conformations mostly present as β‐strands, a broad population of polyproline‐II‐like spirals, and a tight cluster that includes the highly populated α‐helix and the conformationally‐similar but much less populated 310‐helix. Rare, short left‐handed α‐/310‐helical turns with repeating ϕ,ψ‐angles occur, but none are longer than three residues. Misperceptions dispelled by this study are the existence of 2.27‐ and π‐helices as linear groups, the existence of specific ideal ϕ,ψ‐angles for each linear group, and the existence of a substantive difference in the ϕ,ψ‐preferences for parallel versus antiparallel β‐strands. This study provides a concrete basis for updating and enhancing how we think about and teach the basics of protein structure.

Conformational selectivity in cytochrome P450 redox partner interactions

Significance Cytochrome P450 activates molecular oxygen to hydroxylate various molecules. This process requires the transfer of electrons from a redox partner, which further requires formation of a specific protein–protein complex. A question of increasing importance is whether or not the interaction between the P450 and its redox partner results in functionally important structural changes required for activity. The present work addresses the question of whether the redox partner favors binding to a specific conformer. This question is critically important in understanding how P450s activate molecular oxygen. The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein–protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis.

Molecular mechanism of GPCR-mediated arrestin activation

Despite intense interest in discovering drugs that cause G-protein-coupled receptors (GPCRs) to selectively stimulate or block arrestin signalling, the structural mechanism of receptor-mediated arrestin activation remains unclear1,2. Here we reveal this mechanism through extensive atomic-level simulations of arrestin. We find that the receptor’s transmembrane core and cytoplasmic tail—which bind distinct surfaces on arrestin—can each independently stimulate arrestin activation. We confirm this unanticipated role of the receptor core, and the allosteric coupling between these distant surfaces of arrestin, using site-directed fluorescence spectroscopy. The effect of the receptor core on arrestin conformation is mediated primarily by interactions of the intracellular loops of the receptor with the arrestin body, rather than the marked finger-loop rearrangement that is observed upon receptor binding. In the absence of a receptor, arrestin frequently adopts active conformations when its own C-terminal tail is disengaged, which may explain why certain arrestins remain active long after receptor dissociation. Our results, which suggest that diverse receptor binding modes can activate arrestin, provide a structural foundation for the design of functionally selective (‘biased’) GPCR-targeted ligands with desired effects on arrestin signalling.Molecular dynamics simulations and site-directed fluorescence spectroscopy show that the transmembrane core and cytoplasmic tail of G-protein-coupled receptors independently and cooperatively activate arrestin.

Structure-based design of bacterial nitric oxide synthase inhibitors

Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial (Holden, , Proc. Natl. Acad. Sci. U.S.A.2013, 110, 1812724145412). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.

Molecular dynamics of the P450cam–Pdx complex reveals complex stability and novel interface contacts

Cytochrome P450cam catalyzes the stereo and regiospecific hydroxylation of camphor to 5‐exo‐hydroxylcamphor. The two electrons for the oxidation of camphor are provided by putidaredoxin (Pdx), a Fe2S2 containing protein. Two recent crystal structures of the P450cam–Pdx complex, one solved with the aid of covalent cross‐linking and one without, have provided a structural picture of the redox partner interaction. To study the stability of the complex structure and the minor differences between the recent crystal structures, a 100 nanosecond molecular dynamics (MD) simulation of the cross‐linked structure, mutated in silico to wild type and the linker molecule removed, was performed. The complex was stable over the course of the simulation though conformational changes including the movement of the C helix of P450cam further toward Pdx allowed for the formation of a number of new contacts at the complex interface that remained stable throughout the simulation. While several minor crystal contacts were lost in the simulation, all major contacts that had been experimentally studied previously were maintained. The equilibrated MD structure contained a mixture of contacts resembling both the cross‐linked and noncovalent structures and the newly identified interactions. Finally, the reformation of the P450cam Asp251–Arg186 ion pair in the MD simulation mirrors the ion pair observed in the more promiscuous CYP101D1 and suggests that the Asp251–Arg186 ion pair may be important.

P450cin Active Site Water: Implications for Substrate Binding and Solvent Accessibility

In P450cin, Tyr81, Asp241, Asn242, two water molecules, and the substrate participate in a complex H-bonded network. The role of this H-bonded network in substrate binding and catalysis has been probed by crystallography, spectroscopy, kinetics, isothermal titration calorimetry (ITC), and molecular dynamics. For the Y81F mutant, the substrate binds about 20-fold more weakly and Vmax decreases by about 30% in comparison to WT. The enhanced susceptibility of the heme to H₂O₂-mediated destruction in Y81F suggests that this mutant favors the open, low-spin conformational state. Asn242 H-bonds directly with the substrate, and replacing this residue with Ala results in water taking the place of the missing Asn side chain. This mutant exhibits a 70% decrease in activity. Crystal structures and molecular dynamics simulations of substrate-bound complexes show that the solvent has more ready access to the active site, especially for the N242A mutant. This accounts for about a 64% uncoupling of electron transfer from substrate hydroxylation. These data indicate the importance of the interconnected water network on substrate binding and on the open/closed conformational equilibrium, which are both critically important for maintaining high-coupling efficiency.

Crystal structure of cindoxin, the P450cin redox partner.

The crystal structure of the flavin mononucleotide (FMN)-containing redox partner to P450cin, cindoxin (Cdx), has been determined to 1.3 Å resolution. The overall structure is similar to that of the FMN domain of human cytochrome P450 reductase. A Brownian dynamics–molecular dynamics docking method was used to produce a model of Cdx with its redox partner, P450cin. This Cdx–P450cin model highlights the potential importance of Cdx Tyr96 in bridging the FMN and heme cofactors as well P450cin Arg102 and Arg346. Each of the single-site Ala mutants exhibits ∼10% of the wild-type activity, thus demonstrating the importance of these residues for binding and/or electron transfer. In the well-studied P450cam system, redox partner binding stabilizes the open low-spin conformation of P450cam and greatly decreases the stability of the oxy complex. In sharp contrast, Cdx does not shift P450cin to a low-spin state, although the stability of oxy-P450cin is decreased 10-fold in the presence of Cdx. This indicates that Cdx may have a modest effect on the open–closed equilibrium in P450cin compared to that in P450cam. It has been postulated that part of the effector role of Pdx on P450cam is to promote a significant structural change that makes available a proton relay network involving Asp251 required for O2 activation. The structure around the corresponding Asp in P450cin, Asp241, provides a possible structural reason for why P450cin is less dependent on its redox partner for functionally important structural changes.

Elucidating nitric oxide synthase domain interactions by molecular dynamics.

Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing l‐Arg to NO and L‐citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH‐derived electrons are transferred to the heme‐containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN‐to‐heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme–FMN–calmodulin NOS complex based on the recent biophysical studies using a 105‐ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis.

Gi- and Gs-coupled GPCRs show different modes of G-protein binding

Significance Activation of G protein-coupled receptors (GPCRs) initiates conformational shifts that trigger interaction with a specific G-protein subtype from a structurally homologous set. A major unsolved problem is the mechanism by which this selectivity is achieved. Structures of GPCR–G protein complexes so far fail to reveal the origin of selectivity because they all involve one G-protein subtype (Gs). In this work, we report a structural model of the activated GPCR rhodopsin in complex with another G-protein subtype (Gi) derived from intermolecular distance mapping with DEER-EPR and refinement with modeling. Comparison of the model with structures of complexes involving Gs reveals distinct GPCR–G protein-binding modes, the differences of which suggest key features of the structural selectivity filter. More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand–receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR–G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin–G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin–Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR–Gs structures, providing insight into specificity determinants.

Enzymatic Mechanism of Leishmania Major Peroxidase and the Critical Role of Specific Ionic Interactions.

Leishmania major peroxidase (LmP) is very similar to the well-known yeast cytochrome c peroxidase (CcP). Both enzymes catalyze the peroxidation of cytochrome c. Like CcP, LmP reacts with H2O2 to form Compound I, which consists of a ferryl heme and a Trp radical, Fe(IV)═O;Trp(•+). Cytochrome c (Cytc) reduces the Trp radical to give Compound II, Fe(IV)═O;Trp, which is followed by an intramolecular electron transfer to give Fe(III)-OH;Trp(•+), and in the last step, Cytc reduces the Trp radical. In this study, we have used steady-state and single-turnover kinetics to improve our understanding of the overall mechanism of LmP catalysis. While the activity of CcP greatly increases with ionic strength, the kcat for LmP remains relatively constant at all ionic strengths tested. Therefore, unlike CcP, where dissociation of oxidized Cytc is limiting at low ionic strengths, association/dissociation reactions are not limiting at any ionic strength in LmP. We conclude that in LmP, the intramolecular electron transfer reaction, Fe(IV)═O;Trp to Fe(III)-OH;Trp(•+), is limiting at all ionic strengths. Unlike CcP, LmP depends on key intermolecular ion pairs to form the electron transfer competent complex. Mutating these sites causes the initial rate of association to decrease by 2 orders of magnitude and a substantial decrease in kcat. The drop in kcat is due to a switch in the rate-limiting step of the mutants from intramolecular electron transfer to the rate of association in forming the LmP-LmCytc complex. These studies show that while LmP and CcP form very similar complexes and exhibit similar activities, they substantially differ in how their activity changes as a function of ionic strength. This difference is primarily due to the heavy reliance of LmP on highly specific intermolecular ion pairs, while CcP relies mainly on nonpolar interactions.