Relevant Conformations Research Articles

Knowledge-Based Conformer Generation Using the Cambridge Structural Database.

Fast generation of plausible molecular conformations is central to molecular modeling. This paper presents an approach to conformer generation that makes extensive use of the information available in the Cambridge Structural Database. By using geometric distributions derived from the Cambridge Structural Database, it is possible to create biologically relevant conformations in the majority of cases analyzed. The paper compares the performance of the approach with previously published evaluations, and presents some cases where the method fails. The method appears to show significantly improved performance in reproduction of the conformations of structures observed in the Cambridge Structural Database and the Protein Data Bank as compared to other published methods of a similar speed.

Prediction of New Stabilizing Mutations Based on Mechanistic Insights from Markov State Models

Protein stabilization is fundamental to enzyme function and evolution, yet understanding the determinants of a protein's stability remains a challenge. This is largely due to a shortage of atomically-detailed models for the ensemble of relevant protein conformations and their relative populations. For example, the M182T substitution in TEM β-lactamase, an enzyme that confers antibiotic resistance to bacteria, is stabilizing but the precise mechanism remains unclear. Here, we employ Markov state models (MSMs) to uncover how M182T shifts the distribution of different structures that TEM adopts. We find that M182T stabilizes a helix that is a key component of a domain interface. We then predict the effects of other mutations, including a novel stabilizing mutation, and experimentally test our predictions using a combination of stability measurements, crystallography, NMR, and in vivo measurements of bacterial fitness. We expect our insights and methodology to provide a valuable foundation for protein design.

In-cell NMR Spectroscopy for the Investigation of the Conformation of Macromolecules

The non-invasive character of NMR spectroscopy allows researchers to investigate the conformation and dynamics of biological macromolecules in their natural environment, for example the cytoplasm of cells. In the past we have used in-cell NMR investigations to study several proteins as well as the behavior of telomeric DNA in different cellular systems. We will show data on two different systems: The telomeric G-overhang is the 3’ single stranded protrusion of double stranded telomeres and consists of repeating d(TTAGGG)n elements. These elements form G-quadruplexes, which however under different in vitro conditions can adopt several different conformations. In order to investigate which of these conformations is the biologically relevant conformation we have injected quadruplexes of different length into Xenopus oocytes or investigated them in oocytes extracts. These investigations revealed that G4 units coexist in two conformations, the hybrid-2 and the 2-tetrad antiparallel basket. In addition, we have used in-cell NMR to investigate the behavior of Pin-1, a peptidyl-prolyl isomerase and show that the protein uses its WW domain to nonspecifically investigate other proteins as potential substrates. This non-specific interaction can be blocked by phosphorylation in the WW domain.

Structural investigation of cellobiose dehydrogenase IIA: Insights from small angle scattering into intra- and intermolecular electron transfer mechanisms

BackgroundCellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized. MethodsSmall-angle X-ray and neutron scattering measurements of oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa. ResultsThe scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca2+. Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation. ConclusionsThese results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D. General significanceThe present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO.

Pulse EPR distance measurements to study multimers and multimerisation

ABSTRACTPulse dipolar electron paramagnetic resonance (PD-EPR) has become a powerful tool for structural biology determining distances on the nanometre scale. Recent advances in hardware, methodology, and data analysis have widened the scope to complex biological systems. PD-EPR can be applied to systems containing lowly populated conformers or displaying large intrinsic flexibility, making them all but intractable for cryo-electron microscopy and crystallography. Membrane protein applications are of particular interest due to the intrinsic difficulties for obtaining high-resolution structures of all relevant conformations. Many drug targets involved in critical cell functions are multimeric channels or transporters. Here, common approaches for introducing spin labels for PD-EPR cause the presence of more than two electron spins per multimeric complex. This requires careful experimental design to overcome detrimental multi-spin effects and to secure sufficient distance resolution in presence of multiple distances. In addition to obtaining mere distances, PD-EPR can also provide information on multimerisation degrees allowing to study binding equilibria and to determine dissociation constants.

Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.

A novel protein-folding function of RNA has been recognized, which can outperform previously known molecular chaperone proteins. The RNA as a molecular chaperone (chaperna) activity is intrinsic to some ribozymes and is operational during viral infections. Our purpose was to test whether influenza hemagglutinin (HA) can be assembled in a soluble, trimeric, and immunologically activating conformation by means of an RNA molecular chaperone (chaperna) activity. An RNA-interacting domain (RID) from the host being immunized was selected as a docking tag for RNA binding, which served as a transducer for the chaperna function for de novo folding and trimeric assembly of RID-HA1. Mutations that affect tRNA binding greatly increased the soluble aggregation defective in trimer assembly, suggesting that RNA interaction critically controls the kinetic network in the folding/assembly pathway. Immunization of mice resulted in strong hemagglutination inhibition and high titers of a neutralizing antibody, providing sterile protection against a lethal challenge and confirming the immunologically relevant HA conformation. The results may be translated into a rapid response to a new influenza pandemic. The harnessing of the novel chaperna described herein with immunologically tailored antigen-folding functions should serve as a robust prophylactic and diagnostic tool for viral infections.—Yang, S. W., Jang, Y. H., Kwon, S. B., Lee, Y. J., Chae, W., Byun, Y. H., Kim, P., Park, C., Lee, Y. J., Kim, C. K., Kim, Y. S., Choi, S. I., Seong, B. L. Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.

Structure and Dynamics of Immunoglobulin G Glycoproteins.

Immunoglobulin G (IgG) is a major serum glycoprotein that exerts the role of antibody in the immune system. This multifunctional glycoprotein couples antigen recognition with a variety of effector functions promoted via interactions with various IgG-binding proteins. Given its versatile functionality, IgG has recently been used for therapeutic interventions. Evidence indicates that the carbohydrate moieties of IgG glycoproteins critically affect their antibody functions, particularly the effector functions mediated by the interactions with Fcγ receptors (FcγRs). N-glycans at specific positions of FcγR also contribute both positively and negatively to the interactions with IgG. The integration of multilateral biophysical approaches, including X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, has provided structural insights into the mechanisms underlying the glycofunctions of this interacting system. The N-glycans of IgG and FcγR mediate their interactions by either strengthening or weakening the affinity on the basis of their glycoforms. Moreover, the N-glycosylation of IgG-Fc is a prerequisite to maintain the integrity of the quaternary structure of the sites interacting with the effector molecules and can also control functionally relevant local conformations. The biopharmaceutical significance of these glycan functions is discussed from a structural point of view.

Challenges in the Structural-Functional Characterization of Multidomain, Partially Disordered Proteins CBP and p300: Preparing Native Proteins and Developing Nanobody Tools.

The structural and functional characterization of large multidomain signaling proteins containing long disordered linker regions represents special methodological and conceptual challenges. These proteins show extreme structural heterogeneity and have complex posttranslational modification patterns, due to which traditional structural biology techniques provide results that are often difficult to interpret. As demonstrated through the example of two such multidomain proteins, CREB-binding protein (CBP) and its paralogue, p300, even the expression and purification of such proteins are compromised by their extreme proteolytic sensitivity and structural heterogeneity. In this chapter, we describe the effective expression of CBP and p300 in a eukaryotic host, Sf9 insect cells, followed by their tandem affinity purification based on two terminal tags to ensure their structural integrity. The major focus of this chapter is on the development of novel accessory tools, single-domain camelid antibodies (nanobodies), for structural-functional characterization. Specific nanobodies against full-length CBP and p300 can specifically target their different regions and can be used for their marking, labeling, and structural stabilization in a broad range of in vitro and in vivo studies. Here, we describe four high-affinity nanobodies binding to the KIX and the HAT domains, either mimicking known interacting partners or revealing new functionally relevant conformations. As immunization of llamas results in nanobody libraries with a great sequence variation, deep sequencing and interaction analysis with different regions of the proteins provide a novel approach toward developing a panel of specific nanobodies.

An Array-Based Ligand Discovery Platform for Proteins With Short Half-Lives.

Many promising therapeutic protein targets were previously considered "undruggable" due to a deficit in structural information to guide drug design and/or a lack of an obvious binding pocket. Fortunately, array-based methods for evaluating protein binding against large chemical libraries, such as small-molecule microarray screening, have provided one of several emerging inroads to ligand discovery for these elusive targets. Despite the advance in the area of ligand discovery for poorly structured and intrinsically disordered proteins provided by array-based technologies involving cell lysates, the extension of this technology for screening proteins with short half-lives in physiologically relevant conformations has been technically challenging. In this chapter we present a protocol for leveraging in vitro translation strategies to enable array-based screening of short-lived proteins against large small-molecule libraries for ligand discovery.

The influence of polarity in binary solvent mixtures on the conformation of bis-triazinyl-pyridine in solution

ABSTRACTWe report a combined computational and experimental study to investigate the influence of the solvent on the electronic and molecular structure of the bis-triazinyl-pyridine (BTP) ligand. Experimental measurements and quantum-chemical calculations using geometries from molecular dynamics simulations in different solvent methanol/water mixtures reveal a change in the UV/vis absorption spectra for the investigated BTP compound. This change is investigated further using nuclear magnetic resonance (NMR) techniques, both experimental and computational, to gain insight to ligand conformation. Comparison of experimental and computational results enables the analysis of relevant BTP conformers, which cannot be accessed using experimental measurements alone. Based on this approach, we conclude that the BTP ligands change conformation with decreasing solvent polarity to maximise the lipophilic accessible surface – a concept which is transferrable to various classes of compounds.

Modulation of the Neisseria gonorrhoeae drug efflux conduit MtrE

Widespread antibiotic resistance, especially of Gram-negative bacteria, has become a severe concern for human health. Tripartite efflux pumps are one of the major contributors to resistance in Gram-negative pathogens, by efficiently expelling a broad spectrum of antibiotics from the organism. In Neisseria gonorrhoeae, one of the first bacteria for which pan-resistance has been reported, the most expressed efflux complex is MtrCDE. Here we present the electrophysiological characterisation of the outer membrane component MtrE and the membrane fusion protein MtrC, obtained by a combination of planar lipid bilayer recordings and in silico techniques. Our in vitro results show that MtrE can be regulated by periplasmic binding events and that the interaction between MtrE and MtrC is sufficient to stabilize this complex in an open state. In contrast to other efflux conduits, the open complex only displays a slight preference for cations. The maximum conductance we obtain in the in vitro recordings is comparable to that seen in our computational electrophysiology simulations conducted on the MtrE crystal structure, indicating that this state may reflect a physiologically relevant open conformation of MtrE. Our results suggest that the MtrC/E binding interface is an important modulator of MtrE function, which could potentially be targeted by new efflux inhibitors.

Prediction of New Stabilizing Mutations Based on Mechanistic Insights from Markov State Models.

Protein stabilization is fundamental to enzyme function and evolution, yet understanding the determinants of a protein’s stability remains a challenge. This is largely due to a shortage of atomically detailed models for the ensemble of relevant protein conformations and their relative populations. For example, the M182T substitution in TEM β-lactamase, an enzyme that confers antibiotic resistance to bacteria, is stabilizing but the precise mechanism remains unclear. Here, we employ Markov state models (MSMs) to uncover how M182T shifts the distribution of different structures that TEM adopts. We find that M182T stabilizes a helix that is a key component of a domain interface. We then predict the effects of other mutations, including a novel stabilizing mutation, and experimentally test our predictions using a combination of stability measurements, crystallography, NMR, and in vivo measurements of bacterial fitness. We expect our insights and methodology to provide a valuable foundation for protein design.

The activated conformation of integrin β7 is a novel multiple myeloma-specific target for CAR T cell therapy.

Cancer-specific cell-surface antigens are ideal targets for monoclonal antibody (mAb)-based immunotherapy but are likely to have previously been identified in transcriptome or proteome analyses. Here, we show that the active conformer of an integrin can serve as a specific therapeutic target for multiple myeloma (MM). We screened >10,000 anti-MM mAb clones and identified MMG49 as an MM-specific mAb specifically recognizing a subset of integrin β7 molecules. The MMG49 epitope, in the N-terminal region of the β7 chain, is predicted to be inaccessible in the resting integrin conformer but exposed in the active conformation. Elevated expression and constitutive activation of integrin β7 conferred high MMG49 reactivity on MM cells, whereas MMG49 binding was scarcely detectable in other cell types including normal integrin β7+ lymphocytes. T cells transduced with MMG49-derived chimeric antigen receptor (CAR) exerted anti-MM effects without damaging normal hematopoietic cells. Thus, MMG49 CAR T cell therapy is promising for MM, and a receptor protein with a rare but physiologically relevant conformation can serve as a cancer immunotherapy target.

Consequences of Energetic Frustration on the Ligand-Coupled Folding/Dimerization Dynamics of Allosteric Protein S100A12.

Allosteric proteins are featured by energetic degeneracy of two (or more) functionally relevant conformations, therefore their energy landscapes are often locally frustrated. How such frustration affects the protein folding/binding dynamics is not well understood. Here, by using molecular simulations we study the consequences of local frustration in the dimerization dynamics of allosteric proteins based on a homodimer protein S100A12. Despite of the structural symmetry of the two EF-hand motifs in the three-dimensional structures, the S100A12 homodimer shows allosteric behaviors and local frustration only in half of its structural elements, i.e., the C-terminal EF-hand. We showed that such spatially asymmetric location of frustration leads to asymmetric dimerization pathways, in which the dimerization is dominantly initiated by the interchain binding of the minimally frustrated N-terminal EF-hands, achieving optimal balance between the requirements of rapid conformational switching and interchain assembling to the energy landscapes. We also showed that the local frustration, as represented by the double-basin topography of the energy landscape, gives rise to multiple cross-linked dimerization pathways, in which the dimerization is coupled with the allosteric motions of the C-terminal EF-hands. Binding of metal ions tends to reshape the energy landscape and modulate the dimerization pathways. In addition, by employing the frustratometer method, we showed that the highly frustrated residue-pairs in the C-terminal EF-hand are partially unfolded during the conformational transitions of the native homodimer, leading to lowing of free energy barrier. Our results revealed tight interplay between the local frustration of the energy landscape and the dimerization dynamics for allosteric proteins.

Origins of coevolution between residues distant in protein 3D structures

Residue pairs that directly coevolve in protein families are generally close in protein 3D structures. Here we study the exceptions to this general trend-directly coevolving residue pairs that are distant in protein structures-to determine the origins of evolutionary pressure on spatially distant residues and to understand the sources of error in contact-based structure prediction. Over a set of 4,000 protein families, we find that 25% of directly coevolving residue pairs are separated by more than 5 Å in protein structures and 3% by more than 15 Å. The majority (91%) of directly coevolving residue pairs in the 5-15 Å range are found to be in contact in at least one homologous structure-these exceptions arise from structural variation in the family in the region containing the residues. Thirty-five percent of the exceptions greater than 15 Å are at homo-oligomeric interfaces, 19% arise from family structural variation, and 27% are in repeat proteins likely reflecting alignment errors. Of the remaining long-range exceptions (<1% of the total number of coupled pairs), many can be attributed to close interactions in an oligomeric state. Overall, the results suggest that directly coevolving residue pairs not in repeat proteins are spatially proximal in at least one biologically relevant protein conformation within the family; we find little evidence for direct coupling between residues at spatially separated allosteric and functional sites or for increased direct coupling between residue pairs on putative allosteric pathways connecting them.

Modeling Binding with Large Conformational Changes: Key Points in Ensemble-Docking Approaches.

Protein dynamics play a critical role in ligand binding, and different models have been proposed to explain the relationships between protein motion and molecular recognition. Here, we present a study of ligand-binding processes associated with large conformational changes of a protein to elucidate the critical choices in ensemble-docking approaches for effective prediction of the binding geometry. Two study cases were selected in which binding involves different protein motions and intermolecular interactions and, accordingly, conformational selection and induced-fit mechanisms play different roles: binding of multiple ligands to the acetylcholine binding protein and highly specific binding of D-allose to the allose binding protein. Our results indicated that the ensemble-docking technique can provide reliable predictions of the structure of ligand-protein complexes, starting from simulations of the apo systems, when suitable methodological choices are made according to the different mechanistic scenarios. In particular, accelerated molecular dynamics simulations are suitable for conformational sampling when the unbound and bound states are separated by high energy barriers, provided that the acceleration parameters are carefully set to extensively sample the relevant conformations. A strategy specifically developed for geometric clustering of the binding site proved to be effective for selecting a set of conformations relevant to binding from the MD trajectory. Specific strategies have to be selected to incorporate different degrees of ligand-induced protein flexibility into the docking or pose-refinement steps.

Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding.

ExoU is a 74 kDa cytotoxin that undergoes substantial conformational changes as part of its function, that is, it has multiple thermodynamically stable conformations that interchange depending on its environment. Such flexible proteins pose unique challenges to structural biology: (1) not only is it often difficult to determine structures by X-ray crystallography for all biologically relevant conformations because of the flat energy landscape (2) but also experimental conditions can easily perturb the biologically relevant conformation. The first challenge can be overcome by applying orthogonal structural biology techniques that are capable of observing alternative, biologically relevant conformations. The second challenge can be addressed by determining the structure in the same biological state with two independent techniques under different experimental conditions. If both techniques converge to the same structural model, the confidence that an unperturbed biologically relevant conformation is observed increases. To this end, we determine the structure of the C-terminal domain of the effector protein, ExoU, from data obtained by electron paramagnetic resonance spectroscopy in conjunction with site-directed spin labeling and in silico de novo structure determination. Our protocol encompasses a multimodule approach, consisting of low-resolution topology sampling, clustering, and high-resolution refinement. The resulting model was compared with an ExoU model in complex with its chaperone SpcU obtained previously by X-ray crystallography. The two models converged to a minimal RMSD100 of 3.2 Å, providing evidence that the unbound structure of ExoU matches the fold observed in complex with SpcU.

Important issues related to the law of corresponding states for the charge transport in molecular junctions with graphene electrodes

A recent experimental study [Jia et al., Science 352, 1443 (2016)] succeeded to fabricate photoswitches with unprecedented performances using single-molecule junctions based on graphene electrodes and diarylethene that switches between closed and open conformations. The current-voltage measurements reported in that study revealed the applicability of the recently deduced law of corresponding states for the transport by tunneling [Bâldea et al., Nanoscale 7, 10465 (2015)] in a certain range of biases. In the present paper, we demonstrate that the size of that bias range is motivated theoretically. Furthermore, by appropriately extending the theoretical framework, we find an improved agreement between the value of the HOMO energy offset relative to electrodes' Fermi level (which plays the role of a key parameter) extracted from transport data and the value obtained from microscopic calculations. Based on the shape of HOMO spatial distributions, we also present microscopic arguments on why the differences between the two aforementioned estimates for the two relevant (insulating, open and conducting, closed) molecular conformations are different. From a more general perspective, the analysis presented in this study provides important support in favor of a transport mechanism via tunneling in the molecular junctions studied by Jia et al.

Quantum chemical study on phenethylamines reveals new cation structures

Phenethylamines are compounds known by their psychoactive and stimulant effects. Their physiological activity is strongly conformer dependent. In this work, the quantum chemical investigation of neutral and cationic phenylethylamine, amphetamine and methamphetamine was performed in order to determine the most stable physiologically relevant conformers. Calculations were performed employing density functional theory and ωB97XD functional. A previously unknown double-ring cation structure formed by cyclization of aliphatic tail was found for all three molecular species. Calculations performed on several levels of theory reveal significant stability of these double-ring cations relative to other conformers. The transition state structures and energies for the cyclization reactions have been determined. Their stability and structural properties are discussed, as well as the possibility for their experimental observation. The significance of dispersion and NH⋯π interactions between side chain and phenyl ring on stability of phenethylamines and their charged forms were pointed out.

Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding.

Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination.