Triosephosphate Isomerase: N and C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy

Microcrystalline uniformly ¹³C,¹⁵N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.     

Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding

In the N2 domain of the gene-3-protein of phage fd, two consecutive β-strands are connected by a mobile loop of seven residues (157-163). The stability of this loop is low, and the Asp160-Pro161 bond at its tip shows conformational heterogeneity with 90% being in the cis and 10% in the trans form. The refolding kinetics of N2 are complex because the molecules with cis or trans isomers at Pro161 both fold to native-like conformations, albeit with different rates. We employed consensus design to shorten the seven-residue irregular loop around Pro161 to a four-residue type I′ turn without a proline. This increased the conformational stability of N2 by almost 10 kJ mol^− 1 and abolished the complexity of the folding kinetics. Turn sequences obtained from in vitro selections for increased stability strongly resembled those derived from the consensus design. Two other type I′ turns of N2 could also be stabilized by consensus design. For all three turns, the gain in stability originates from an increase in the rate of refolding. The turns form native-like structures early during refolding and thus stabilize the folding transition state. The crystal structure of the variant with all three stabilized turns confirms that the 157-163 loop was in fact shortened to a type I′ turn and that the other turns maintained their type I′ conformation after sequence optimization.     

Crystal Structure of Human RNA Helicase A (DHX9): Structural Basis for Unselective Nucleotide Base Binding in a DEAD-Box Variant Protein

RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long α-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5′-triphosphate, cytidine 5′-triphosphate, and uridine 5′-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.     

Crystal Structures of Inhibitor Complexes of Human T-Cell Leukemia Virus (HTLV-1) Protease

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.     

Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

The ΔF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and ΔF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because ΔF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and ΔF508 constructs, and the ΔF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide ¹H/²H exchange rates in matched F508 and ΔF508 constructs reveal that ΔF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the ΔF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-ΔF508 structures but completely solvent exposed in all ΔF508 structures. These results reinforce the importance of the perturbation ΔF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.     

Crystal Structures of Penicillin-Binding Proteins 4 and 5 from Haemophilus influenzae

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel β-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by β-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position.     

Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium

The crystal structure of Bifidobacterium longum phosphoketolase, a thiamine diphosphate (TPP) dependent enzyme, has been determined at 2.2 Å resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits with molecular mass of 92.5 kDa. The bound TPP is almost completely shielded from solvent except for the catalytically important C2-carbon of the thiazolium ring, which can be accessed by a substrate sugar through a narrow funnel-shaped channel. In silico docking studies of B. longum phosphoketolase with its substrate enable us to propose a model for substrate binding.

Structured summary

MINT-7985878: PKT (uniprotkb:Q6R2Q7) and PKT (uniprotkb:Q6R2Q7) bind (MI:0407) by X-ray crystallography (MI:0114)     

Structure of a polyisoprenoid binding domain from Saccharophagus degradans implicated in plant cell wall breakdown

Saccharophagus degradans belongs to a recently discovered group of marine bacteria equipped with an arsenal of sugar cleaving enzymes coupled to carbohydrate-binding domains to degrade various insoluble complex polysaccharides. The modular Sde-1182 protein consists of a family 2 carbohydrate binding module linked to a X158 domain of unknown function. The 1.9 Å and 1.55 Å resolution crystal structures of the isolated X158 domain bound to the two related polyisoprenoid molecules, ubiquinone and octaprenyl pyrophosphate, unveil a β-barrel architecture reminiscent of the YceI-like superfamily that resembles the architecture of the lipocalin fold. This unprecedented association coupling oxidoreduction and carbohydrate recognition events may have implications for effective nutrient uptake in the marine environment.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

On the relationship between the activity and structure of PEG-alpha-chymotrypsin conjugates in organic solvents

Enzymes are attractive catalysts for the production of optically active compounds in organic solvents. However, their often low catalytic activity in such applications hampers their practical use. To overcome this, we investigated the effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) with a Mw of 5,000 to enhance its activity. The model transesterification reaction between sec-phenethyl alcohol and vinyl butyrate in various neat dry organic solvents and at a controlled water activity of 0.008 in two solvents was employed to measure the effect of PEGylation on activity and enantioselectivity. Synthesis conditions were varied to obtain various conjugates with average molar ratios of PEG-to-chymotrypsin ranging from ca. 1 to 7. While the enantioselectivity increased only modestly from ca. 4.4 to 6.1 when averaging results in all solvents, PEG was very efficient in increasing the activity of alpha-chymotrypsin up to more than 400-fold compared to that of the powder lyophilized from buffer alone. The activity increase was more pronounced in apolar than in polar organic solvents and also depended on the amount of PEG bound to the enzyme. For example, the activity of the modified enzyme towards the most reactive "S" enantiomer in octane increased 440-fold but increasing the molar ratio of PEG-to-enzyme from 1.1 to 7.1 resulted in a more than twofold decrease in enzyme activity. Controlling the water activity did not prevent the drop in activity. To investigate the possible origin of the activity changes, Fourier transform infrared (FTIR) spectroscopy experiments were conducted. It was found that PEGylation reduced lyophilization-induced structural perturbations, but exposure to the organic solvents caused structural perturbations. These perturbations were more pronounced in polar than in apolar solvents. The pronounced activity drop in polar solvents at increasing PEG-modification levels correlated with an increasing level of solvent-induced structural perturbations. This correlation was less pronounced in apolar solvents where both, activity drop and structural perturbations, were less pronounced at increasing PEGylation levels. In summary, PEG-modified alpha-chymotrypsin might be an interesting system to catalyze reactions, particularly in apolar organic solvents.