Direct coordination of pterin to FeII enables neurotransmitter biosynthesis in the pterin-dependent hydroxylases.

The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the FeII active site to form a highly reactive FeIV = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-FeIV = O intermediate and characterized its structure as a FeII-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate-bound ternary FeII active site before the O2 reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of FeIV = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the FeII site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the FeII site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes.

Templated Enzymatic Synthesis of δ-Cyclodextrin.

While α-, ß-, and γ-cyclodextrin (CD) are ubiquitous hosts employed by supramolecular chemists, δ-CD (formed from nine α-1,4-linked glucopyranose units) has received very little attention. α-, ß-, and γ-CD are the major products of the enzymatic breakdown of starch by cyclodextrin glucanotransferase (CGTase), but δ-CD forms only transiently in this reaction, as a minor component of a complex mixture of linear and cyclic glucans. In this work, we show how δ-CD can be synthesized in unprecedented yields by employing a bolaamphiphile template in an enzyme-mediated dynamic combinatorial library of cyclodextrins. NMR spectroscopy studies revealed that δ-CD can thread up to three bolaamphiphiles forming [2]-, [3]-, or [4]-pseudorotaxanes, depending on the size of the hydrophilic headgroup and the length of the alkyl chain axle. Threading of the first bolaamphiphile occurs in fast exchange on the NMR chemical shift time scale, while subsequent threading occurs in slow exchange. To extract quantitative information for 1:2 and 1:3 binding events occurring in mixed exchange regimes, we derived equations for nonlinear curve fitting that take into consideration both the chemical shift changes for species in fast exchange and the integrals for species in slow exchange to determine Ka1, Ka2, and Ka3. Template T1 could be used to direct the enzymatic synthesis of δ-CD due to the cooperative formation of a 1:2 complex─the [3]-pseudorotaxane δ-CD·T12. Importantly, T1 is recyclable. It can be readily recovered from the enzymatic reaction by precipitation and reused in subsequent syntheses enabling preparative-scale synthesis of δ-CD.

Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate-dependent nonheme ferrous enzymes.

Determining the requirements for efficient oxygen (O2) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O2) and a sequential mechanism (cofactor binding and reaction with O2 precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the FeII), αKG binding to FeII-DAOCS results in ∼45% five-coordinate sites that selectively react with O2 relative to the remaining six-coordinate sites. However, this reaction produces an FeIII species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to FeII-DAOCS produces five-coordinate sites that rapidly react with O2 to form an FeIV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes.

Substrates and Cyclic Peptide Inhibitors of the Oligonucleotide-Activated Sirtuin 7.

The sirtuins are NAD+ -dependent lysine deacylases, comprising seven isoforms (SIRT1-7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ-N-acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small-angle X-ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism-based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA-display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18.

Dynamics of Human Serum Transferrin in Varying Physicochemical Conditions Explored by Using Molecular Dynamics Simulations.

Conformational stability of human serum transferrin (Tf) at varying pH values and salt and excipient concentrations were investigated using molecular dynamics (MD) simulations, and the results are compared with previously published small-angle X-ray scattering (SAXS) experiments. SAXS study showed that at pH 5, Tf is predominantly present in a partially open (PO) form, and the factions of PO differ based on the physicochemical condition and drift toward the closed form (HO) as the pH increases. Tf is a bilobal glycoprotein that is composed of homologous halves termed the N- and C-lobes. The current study shows that the protonation of Y188 and K206 at pH 5 is the primary conformational drive into PO, which shifts toward the closed (HO) conformer as the pH increases. Furthermore, at pH 6.5, PO is unfavorable due to negative charge-charge repulsion at the N/C-lobe interface linker region causing increased hinge distance when compared to HO, which has favorable attractive electrostatic interactions in this region. Subsequently, the effect of salt concentration was studied at 70 and 140 mM NaCl. At 70 mM NaCl and pH 5, chloride ions bind strongly in the N-lobe iron-binding site, whereas these interactions are weak at pH 6.5. With increasing salt concentration at pH 5, the regions surrounding the N-lobe iron-binding site are saturated, and as a consequence, sodium and chloride ions accumulate into the bulk. Additionally, protein-excipient interactions were investigated. At pH 5, the excipients interact in similar loop regions, E89-T93, and D416-D420, located in the N- and C-lobes of the HO conformer, respectively. It is anticipated that interactions of additives in these two loop regions cause conformational changes that lead to iron-coordinating residues in the N-lobe to drift away from iron and thus drive HO to PO conversion. Furthermore, at pH 6.5 and 140 mM histidine, these interactions are negligible leading to the stabilization of HO.

Self-Interactions of Two Monoclonal Antibodies: Small-Angle X-ray Scattering, Light Scattering, and Coarse-Grained Modeling.

Using light scattering (LS), small-angle X-ray scattering (SAXS), and coarse-grained Monte Carlo (MC) simulations, we studied the self-interactions of two monoclonal antibodies (mAbs), PPI03 and PPI13. With LS measurements, we obtained the osmotic second virial coefficient, B22, and the molecular weight, Mw, of the two mAbs, while with SAXS measurements, we studied the mAbs' self-interaction behavior in the high protein concentration regime up to 125 g/L. Through SAXS-derived coarse-grained representations of the mAbs, we performed MC simulations with either a one-protein or a two-protein model to predict B22. By comparing simulation and experimental results, we validated our models and obtained insights into the mAbs' self-interaction properties, highlighting the role of both ion binding and charged patches on the mAb surfaces. Our models provide useful information about mAbs' self-interaction properties and can assist the screening of conditions driving to colloidal stability.

Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus.

Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked ß-d-mannuronic acid (M) and α-l-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel ß-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel ß-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.

Water Distribution and Clustering on the Lyophilized IgG1 Surface: Insight from Molecular Dynamics Simulations.

Water has a critical role in the stability of the higher-order structure of proteins. In addition, it is considered to be a major destabilization factor for the physical and chemical stability of freeze-dried proteins and peptides. Physical and chemical aspects of protein/water relationships are commonly studied with the use of water vapor sorption isotherms for amorphous lyophilized proteins, which, in turn, are commonly analyzed using the Brunauer-Emmett-Teller (BET) equation to obtain the parameters, Wm and CB. The parameter Wm is generally referred to as the "monolayer limit of adsorption" and has a narrow range of 6-8% for most proteins. In this study, the water distribution on an IgG1 surface is investigated by molecular dynamics (MD) simulations at different water contents. The monolayer of water molecules was found to have limited coverage of the protein surface, and the true monolayer coverage of the protein globule actually occurs at a hydration level above 30%. The distribution of water molecules on the IgG1 surface is also highly heterogeneous, and the heterogeneity is not considered in the BET theory. In this study, a mechanistic model has been developed to describe the water vapor sorption isotherm. This model is based on the analysis of the hydrogen bonding network extracted from the MD simulations. The model is consistent with the experimental Type-II isotherm, which is usually observed for proteins. The physical meaning of the BET monolayer was redefined as the onset of water cluster formation. A simple model to calculate the onset water level, Wm, is proposed based on the hydration of different amino acids, as determined from the MD simulations.

Investigations of Albumin-Insulin Detemir Complexes Using Molecular Dynamics Simulations and Free Energy Calculations.

Insulin detemir is a lipidated insulin analogue that obtains a half-life extension by oligomerization and reversible binding to human serum albumin. In the present study, the complex between a detemir hexamer and albumin is investigated by an integrative approach combining molecular dynamics (MD) simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculations, and dynamic light scattering (DLS) experiments. Recent reported small-angle X-ray scattering data could not unambiguously resolve the exact binding site of detemir on albumin. We therefore applied MD simulations to deduce the binding site and key protein-protein interactions. MD simulations were started from initial complex structures based on the SAXS models, and free energies of binding were estimated from the simulations by using the MM-PBSA approach for the different binding positions. The results suggest that the overlapping FA3-FA4 binding site (named FA4) is the most favorable site with a calculated free energy of binding of -28 ± 6 kcal/mol and a good fit to the reported SAXS data throughout the simulations. Multiple salt bridges, hydrogen bonds, and favorable van der Waals interactions are observed in the binding interface that promote complexation. The binding to FA4 is further supported by DLS competition experiments with the prototypical FA4 ligand, ibuprofen, showing displacement of detemir by ibuprofen. This study provides information on albumin-detemir binding on a molecular level, which could be utilized in a rational design of future lipidated albumin-binding peptides.

The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening.

Therapeutic peptides and proteins show enormous potential in the pharmaceutical market, but high costs in discovery and development are limiting factors so far. Single or multiple point mutations are commonly introduced in protein drugs to increase their binding affinity or selectivity. They can also induce adverse properties, which might be overlooked in a functional screen, such as a decreased colloidal or thermal stability, leading to problems in later stages of the development. In this study, we address the effect of point mutations on the stability of the 4.4 kDa antimicrobial peptide plectasin, as a case study. We combined a systematic high-throughput biophysical screen of the peptide thermal and colloidal stability using dynamic light scattering and differential scanning calorimetry with structure-based methods including small-angle X-ray scattering, analytical ultracentrifugation, and nuclear magnetic resonance spectroscopy. Additionally, we applied molecular dynamics simulations to link obtained protein stability parameters to the protein's molecular structure. Despite their predicted structural similarities, all four plectasin variants showed substantially different behavior in solution. We observed an increasing propensity of plectasin to aggregate at a higher pH, and the introduced mutations influenced the type of aggregation. Our strategy for systematically assessing the stability and aggregation of protein drugs is generally applicable and is of particular relevance, given the increasing number of protein drugs in development.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life.

Glucagon-like peptide 1 (GLP-1) is a small incretin hormone stimulated by food intake, resulting in an amplification of the insulin response. Though GLP-1 is interesting as a drug candidate for the treatment of type 2 diabetes mellitus, its short plasma half-life of <3 min limits its clinical use. A strategy for extending the half-life of GLP-1 utilizes the long half-life of human serum albumin (HSA) by combining the two via chemical conjugation or genetic fusion. HSA has a plasma half-life of around 21 days because of its interaction with the neonatal Fc receptor (FcRn) expressed in endothelial cells of blood vessels, which rescues circulating HSA from lysosomal degradation. We have conjugated GLP-1 to C34 of native sequence recombinant HSA (rHSA) and two rHSA variants, one with increased and one with decreased binding affinity for human FcRn. We have investigated the impact of conjugation on FcRn binding affinities, GLP-1 potency, and pharmacokinetics, combined with the solution structure of the rHSA variants and GLP-1-albumin conjugates. The solution structures, determined by small-angle X-ray scattering, show the GLP-1 pointing away from the surface of rHSA. Combining the solution structures with the available structural information about the FcRn and GLP-1 receptor obtained from X-ray crystallography, we can explain the observed in vitro and in vivo behavior. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease in the potency of GLP-1 can be explained by a steric hindrance of binding of GLP-1 to its receptor.

Revealing the Compact Structure of Lactic Acid Bacterial Heteroexopolysaccharides by SAXS and DLS.

Molecular structures of exopolysaccharides are required to understand their functions and the relationships between the structure and physical and rheological properties. Small-angle X-ray scattering and dynamic light scattering were used in conjunction with molecular modeling to characterize solution structures of three lactic acid bacterial heteroexopolysaccharides (HePS-1, HePS-2, and HePS-3). Values of radius of gyration RG, cross-sectional radius of gyration RXS, approximate length L, and hydrodynamic diameter were not directly proportional to the molar mass and indicated the HePSs adopted a compact coil-like rather than an extended conformation. Constrained molecular modeling of 15000 randomized HePS-1 conformers resulted in five best-fit structures with R factor of 3.9-4.6% revealing random coil-like structure. Φ and Ψ angle analysis of glycosidic linkages in HePS-1 structures suggests Galf residues significantly influence the conformation. Ab initio scattering modeling of HePS-2 and HePS-3 gave excellent curve fittings with χ2 of 0.43 and 0.34 for best-fit models, respectively, compatible with coil-like conformation. The findings disclose solution behavior of HePS relevant for their interactions with biomacromolecules, for example, milk proteins.

Small-Angle X-ray Scattering Data in Combination with RosettaDock Improves the Docking Energy Landscape.

We have performed a benchmark to evaluate the relative success of using small-angle X-ray scattering (SAXS) data as constraints (hereafter termed SAXSconstrain) in the RosettaDock protocol (hereafter termed RosettaDockSAXS). For this purpose, we have chosen 38 protein complex structures, calculated the theoretical SAXS data for the protein complexes using the program CRYSOL, and then used the SAXS data as constraints. We further considered a few examples where crystal structures and experimental SAXS data are available. SAXSconstrain were added to the protocol in the initial, low-resolution docking step, allowing fast rejection of complexes that violate the shape restraints imposed by the SAXS data. Our results indicate that the implementation of SAXSconstrain in general reduces the sampling space of possible protein-protein complexes significantly and can indeed increase the probability of finding near-native protein complexes. The methodology used is based on rigid-body docking and works for cases where no or minor conformational changes occur upon binding of the docking partner. In a wider perspective, the strength of RosettaDockSAXS lies in the combination of low-resolution structural information on protein complexes in solution from SAXS experiments with protein-protein interaction energies obtained from RosettaDock, which will allow the prediction of unknown three-dimensional atomic structures of protein-protein complexes.

In-silico study of the interactions between acylated glucagon like-peptide-1 analogues and the native receptor.

We have performed a series of multiple molecular dynamics (MD) simulations of glucagon-like peptide-1 (GLP-1) and acylated GLP-1 analogues in complex with the endogenous receptor (GLP-1R) to obtain a molecular understanding of how fatty acid (FA) chain structure, acylation position on the peptide, and presence of a linker affect the binding. MD simulations were analysed to extract heatmaps of receptor-peptide interaction patterns and to determine the free energy of binding using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach. The extracted free energies from MM-PBSA calculations are in qualitative agreement with experimentally determined potencies. Furthermore, the interaction patterns seen in the receptor-GLP-1 complex simulations resemble previously reported binding interactions validating the simulations. Analysing the receptor-GLP-1 analogue complex simulations, we found that the major differences between the systems stem from FA interactions and positioning of acylation in the peptide. Hydrophobic interactions between the FA chain and a hydrophobic patch on the extracellular domain contribute significantly to the binding affinity. Acylation on Lys26 resulted in noticeably more interactions between the FA chain and the extracellular domain hydrophobic patch than found for acylation on Lys34 and Lys38, respectively. The presence of a charged linker between the peptide and FA chain can potentially stabilise the complex by forming hydrogen bonds to arginine residues in the linker region between the extracellular domain and the transmembrane domain. A molecular understanding of the fatty acid structure and its effect on binding provides important insights into designing acylated agonists for GLP-1R.Communicated by Ramaswamy H. Sarma.

Combining Molecular Dynamics Simulations and Biophysical Characterization to Investigate Protein-Specific Excipient Effects on Reteplase during Freeze Drying.

We performed molecular dynamics simulations of Reteplase in the presence of different excipients to study the stabilizing mechanisms and to identify the role of excipients during freeze drying. To simulate the freeze-drying process, we divided the process into five distinct steps: (i) protein-excipient formulations at room temperature, (ii) the ice-growth process, (iii)-(iv) the partially solvated and fully dried formulations, and (v) the reconstitution. Furthermore, coarse-grained (CG) simulations were employed to explore the protein-aggregation process in the presence of arginine. By using a coarse-grained representation, we could observe the collective behavior and interactions between protein molecules during the aggregation process. The CG simulations revealed that the presence of arginine prevented intermolecular interactions of the catalytic domain of Reteplase, thus reducing the aggregation propensity. This suggests that arginine played a stabilizing role by interacting with protein-specific regions. From the freeze-drying simulations, we could identify several protein-specific events: (i) collapse of the domain structure, (ii) recovery of the drying-induced damages during reconstitution, and (iii) stabilization of the local aggregation-prone region via direct interactions with excipients. Complementary to the simulations, we employed nanoDSF, size-exclusion chromatography, and CD spectroscopy to investigate the effect of the freeze-drying process on the protein structure and stability.

Candida antarctica lipase B performance in organic solvent at varying water activities studied by molecular dynamics simulations.

Applications of lipases in low-water environments are found across a broad range of industries, including the pharmaceutical and oleochemical sectors. This includes condensation reactions in organic solvents where the enzyme activity has been found to depend strongly on both the solvent and the water activity (aw). Despite several experimental and computational studies, knowledge is largely empirical, and a general predictive approach is much needed. To close this gap, we chose native Candida antarctica lipase B (CALB) and two mutants thereof and used molecular dynamics (MD) simulations to gain a molecular understanding of the effect of aw on the specific activity of CALB in hexane. Based on the simulations, we propose four criteria to understand the performance of CALB in organic media, which is supported by enzyme kinetics experiments. First, the lipase must be stable in the organic solvent, which was the case for native CALB and the two mutants studied here. Secondly, water clusters that form and grow close to the active site must not block the path of substrate molecules into the active site. Thirdly, the lipase's lid must not cover the active site. Finally, mutations and changes in aw must not disrupt the geometry of the active site. We show that mutating specific residues close to the active site can hinder water cluster formation and growth, making the lipase resistant to changes in aw. Our computational screening criteria could potentially be used to screen in-silico designed variants, so only promising candidates could be pushed forward to characterisation.

Molecular insights into alginate ß-lactoglobulin A multivalencies-The foundation for their amorphous aggregates and coacervation.

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of ß-lactoglobulin A (ß-LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. ß-LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on ß-LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to ß-LgA, as concluded from saturation transfer difference and 1 H-ligand-targeted NMR analyses. Molecular docking using Glide within the Schrödinger suite 2016-1 revealed the orientation of AOSs to only vary slightly at the preferred ß-LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the ß-LgA complexes may help explain the first steps toward disordered ß-LgA alginate coacervate structures.

Structural characterization of human tryptophan hydroxylase 2 reveals that L-Phe is superior to L-Trp as the regulatory domain ligand.

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the brain. Consequently, regulation of TPH2 is relevant for serotonin-related diseases, yet the regulatory mechanism of TPH2 is poorly understood and structural and dynamical insights are missing. We use NMR spectroscopy to determine the structure of a 47 N-terminally truncated variant of the regulatory domain (RD) dimer of human TPH2 in complex with L-Phe, and show that L-Phe is the superior RD ligand compared with the natural substrate, L-Trp. Using cryo-EM, we obtain a low-resolution structure of a similarly truncated variant of the complete tetrameric enzyme with dimerized RDs. The cryo-EM two-dimensional (2D) class averages additionally indicate that the RDs are dynamic in the tetramer and likely exist in a monomer-dimer equilibrium. Our results provide structural information on the RD as an isolated domain and in the TPH2 tetramer, which will facilitate future elucidation of TPH2's regulatory mechanism.