Zinc-induced Self-association of Complement C3b and Factor H: IMPLICATIONS FOR INFLAMMATION AND AGE-RELATED MACULAR DEGENERATION*

The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100 μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions showed that putative weak zinc binding sites with different capacities exist in all five proteins, in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much reduced at 60 μm zinc and even more so at >100 μm zinc. The removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced age-related macular degeneration in higher risk patients.     

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into ¹⁵N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn¹¹, Arg¹³, Ala²², Arg²⁸, Ser³², Arg³⁶, Lys⁴¹, Lys⁵⁷, Tyr⁶⁴, Lys⁶⁷, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg¹³, Tyr¹⁶, Arg²⁸, Ser⁴², Lys⁴⁸, Lys⁵⁰, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K_d values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.     

Improvement in the oxidative folding of endothelin-1 by a Lys-Arg extension at the amino terminus: Implication of a salt bridge between Arg-1 and Asp8

An amino-terminal extension of endothelin-1 by the Lys-Arg dipeptide in the prosequence (KR-ET-1) greatly increased the ratio of native-type to non-native-type disulfide isomer (96/4 versus 71/29) during the oxidative folding reaction. This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of it was maintained with similar carboxamide analogues replaced at Glu¹⁰ or Asp¹⁸. Structure analyses by circular dichroism spectroscopy revealed that (i) in the carboxylate state, the -helical content of the native-type isomer of KR-ET-1 is higher than that of the native-type isomer of ET-1, while such a variation is not observed in the corresponding non-native-type isomer of KR-ET-1; and (ii) the enhanced -helicity resulting from the Lys-Arg extension is largely diminished in D8N-KR-ET-1. From these results and our previous findings that the helical structure in KR-ET-1 is stabilized by a particular salt bridge between the extended Arg^-1 basic moiety and either the Asp⁸ or Glu¹⁰ acidic side chain in ET-1 [Aumelas, A. et al., Biochemistry, 34 (1995) 4546], we conclude that the formation of a specific salt bridge between the side chains of Arg^-1 and Asp⁸ in KR-ET-1 is critical for the predominant generation of the native-type disulfide isomer, probably because it stabilizes the helical structure of parental ET-1.     

C3larvin Toxin,an ADP-ribosyltransferase from Paenibacillus larvae

C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD⁺ (K_m = 34 ± 12 μm) and RhoA (K_m = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (K_i = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins.     

Enhancing Integrin α1 Inserted (I) Domain Affinity to Ligand Potentiates Integrin α1β1-mediated Down-regulation of Collagen Synthesis

Integrin α1β1 binding to collagen IV, which is mediated by the α1-inserted (I) domain, down-regulates collagen synthesis. When unligated, a salt bridge between Arg²⁸⁷ and Glu³¹⁷ is thought to keep this domain in a low affinity conformation. Ligand binding opens the salt bridge leading to a high-affinity conformation. How modulating integrin α1β1 affinity alters collagen homeostasis is unknown. To address this question, we utilized a thermolysin-derived product of the α1α2α1 network of collagen IV (α1α2α1(IV) truncated protomer) that selectively binds integrin α1β1. We show that an E317A substitution enhanced binding to the truncated protomer, consistent with a previous finding that this substitution eliminates the salt bridge. Surprisingly, we show that an R287A substitution did not alter binding, whereas R287E/E317R substitutions enhanced binding to the truncated protomer. NMR spectroscopy and molecular modeling suggested that eliminating the Glu³¹⁷ negative charge is sufficient to induce a conformational change toward the open state. Thus, the role played by Glu³¹⁷ is largely independent of the salt bridge. We further show that cells expressing E317A or R287E/E317R substitutions have enhanced down-regulation of collagen IV synthesis, which is mediated by the ERK/MAPK pathway. In conclusion, we have demonstrated that modulating the affinity of the extracellular α1 I domain to collagen IV enhances outside-in signaling by potentiating ERK activation and enhancing the down-regulation of collagen synthesis.     

A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter

The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people. Here we examined the contribution of the C terminus in supporting folding of the serotonin transporter (SERT; SLC6A4). Our working hypothesis posited that the amphipathic nature of the C-terminal α-helix (Thr⁶⁰³–Thr⁶¹³) was important for folding of SERT. Accordingly, we disrupted the hydrophobic moment of the α-helix by replacing Phe⁶⁰⁴, Ile⁶⁰⁸, or Ile⁶¹² by Gln. The bulk of the resulting mutants SERT-F604Q, SERT-I608Q, and SERT-I612Q were retained in the endoplasmic reticulum, but their residual delivery to the cell surface still depended on SEC24C. This indicates that the amphipathic nature of the C-terminal α-helix was dispensable to endoplasmic reticulum export. The folding trajectory of SERT is thought to proceed through the inward facing conformation. Consistent with this conjecture, cell surface expression of the misfolded mutants was restored by (i) introducing second site suppressor mutations, which trap SERT in the inward facing state, or (ii) by the pharmacochaperone noribogaine, which binds to the inward facing conformation. Finally, mutation of Glu⁶¹⁵ at the end of the C-terminal α-helix to Lys reduced surface expression of SERT-E615K. A charge reversal mutation in intracellular loop 1 restored surface expression of SERT-R152E/E615K to wild type levels. These observations support a mechanistic model where the C-terminal amphipathic helix is stabilized by an intramolecular salt bridge between residues Glu⁶¹⁵ and Arg¹⁵². This interaction acts as a pivot in the conformational search associated with folding of SERT.     

Structure and dynamics of α‐MSH using DRISM integral equation theory and stochastic dynamics

The structural and dynamical features of the hormone α‐MSH in solution have been examined over a 100 ns time scale by using free energy molecular mechanics models at room temperature. The free energy surface has been modeled using methods from integral equation theory and the dynamics by the Langevin equation. A modification of the accessible surface area friction drag model was used to calculate the atomic friction coefficients. The molecule shows a stable β‐turn conformation in the message region and a close interaction between the side chains of His⁶, Phe⁷, and Trp⁹. A salt bridge between Glu⁵ and Arg⁸ was found not to be a preferred interaction, whereas a Glu⁵ and Lys¹¹ salt bridge was not sampled, presumably due to relatively high free energy barriers. The message region was more conformationally rigid than the N‐terminal region. Several structural features observed here agree well with experimental results. The conformational features suggest a receptor–hormone interaction model where the hydrophobic side chains of Phe⁷ and Trp⁹ interact with the transmembrane portion of the MC1 receptor. Also, the positively charged side chain of Arg⁸ and the imidazole side chain of His⁶ may interact with the negatively charged portions of the receptor which may even be on the receptor's extracellular loops. © 1999 John Wiley & Sons, Inc. Biopoly 50: 255–272, 1999     

Improvement in the oxidative folding of endothelin-1 by a Lys-Arg extension at the amino terminus: Implication of a salt bridge between Arg-1 and Asp8

Summary An amino-terminal extension of endothelin-l by the lys-Arg dipeptide in the prosequence (KR-ET-1) greatly increased the ratio of native-type to non-native-type disulfide isomer (96/4 versus 71/29) during the oxidative folding reaction. This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of it was maintained with similar carboxamide analogues replaced at Glu¹⁰ or Asp¹⁸. Structure analyses by circular dichroism spectroscopy revealed that (i) in the carboxylate state, the α-helical content of the native-type isomer of KR-ET-l is higher than that of the native-type isomer of ET-1, while such a variation is not observed in the corresponding non-native-type isomer of KR-ET-l; and (ii) the enhanced α-helicity resulting from the Lys-Arg extension is largely diminished in D8N-KR-ET-l. From these results and our previous findings that the helical structure in KR-ET-l is stabilized by a particular salt bridge between the extended Arg⁻¹ basic moiety and either the Asp⁸ or Glu¹⁰ acidic side chain in Et-1 [Aumelas, A. et al., Biochemistry, 34 (1995) 4546], we conclude that the formation of a specific salt bridge between the side chains of Arg⁻¹ and Asp⁸ in KR-ET-1 is critical for the predominant generation of the native-type disulfide isomer, probably because it stabilizes the helical structure of parental ET-1.     

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg⁵⁰⁶ and Arg³⁰⁶ and of factor VIIIa (FVIIIa) by cleavage at Arg³³⁶ and Arg⁵⁶². To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K_D of 0.11 ± 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg⁵⁰⁶ and not Arg³⁰⁶ and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg⁵⁰⁶ cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg⁵⁰⁶ interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.     

Subunit Symmetry at the Extracellular Domain-Transmembrane Domain Interface in Acetylcholine Receptor Channel Gating

Transmitter molecules bind to synaptic acetylcholine receptor channels (AChRs) to promote a global channel-opening conformational change. Although the detailed mechanism that links ligand binding and channel gating is uncertain, the energy changes caused by mutations appear to be more symmetrical between subunits in the transmembrane domain compared with the extracellular domain. The only covalent connection between these domains is the pre-M1 linker, a stretch of five amino acids that joins strand β10 with the M1 helix. In each subunit, this linker has a central Arg (Arg^3′), which only in the non-α-subunits is flanked by positively charged residues. Previous studies showed that mutations of Arg^3′ in the α-subunit alter the gating equilibrium constant and reduce channel expression. We recorded single-channel currents and estimated the gating rate and equilibrium constants of adult mouse AChRs with mutations at the pre-M1 linker and the nearby residue Glu⁴⁵ in non-α-subunits. In all subunits, mutations of Arg^3′ had similar effects as in the α-subunit. In the ϵ-subunit, mutations of the flanking residues and Glu⁴⁵ had only small effects, and there was no energy coupling between ϵGlu⁴⁵ and ϵArg^3′. The non-α-subunit Arg^3′ residues had Φ-values that were similar to those for the α-subunit. The results suggest that there is a general symmetry between the AChR subunits during gating isomerization in this linker and that the central Arg is involved in expression more so than gating. The energy transfer through the AChR during gating appears to mainly involve Glu⁴⁵, but only in the α-subunits.     

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)₈-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp²⁴¹, Glu²⁹⁵, Asp³⁶⁹, His¹⁴⁵, and His³⁶⁸) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a K_m of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe³⁺ and Hg²⁺ and was enhanced by Mn²⁺ and Mg²⁺. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu⁴⁹⁸ and Arg³¹⁰ with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.     

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2_hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2_hPg. However, the binding strength of VEK30 (K_D = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (K_D = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH₂ and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2_hPg with nearly the same affinity as PAM (K_D = 1–2 nm). However, addition of two PAM residues (Arg¹²⁶-His¹²⁷) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2_hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg¹¹³, His¹¹⁴, Glu¹¹⁶, Arg¹²⁶, His¹²⁷), mutation of which reduced PAM binding affinity for K2_hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

The Fab Conformations in the Solution Structure of Human Immunoglobulin G4 (IgG4) Restrict Access to Its Fc Region: IMPLICATIONS FOR FUNCTIONAL ACTIVITY*

Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser²²²) and a hinge mutant IgG4(Pro²²²) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s_20,w⁰ of 6.6–6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration R_g was unchanged with concentration in 50–250 mm NaCl buffers, whereas the neutron R_g values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser²²²) with extended hinge structures. The IgG4(Pro²²²) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications.     

Exploring Strong Interactions in Proteins with Quantum Chemistry and Examples of Their Applications in Drug Design

Objectives

Three strong interactions between amino acid side chains (salt bridge, cation-π, and amide bridge) are studied that are stronger than (or comparable to) the common hydrogen bond interactions, and play important roles in protein-protein interactions.

Methods

Quantum chemical methods MP2 and CCSD(T) are used in calculations of interaction energies and structural optimizations.

Results

The energies of three types of amino acid side chain interactions in gaseous phase and in aqueous solutions are calculated using high level quantum chemical methods and basis sets. Typical examples of amino acid salt bridge, cation-π, and amide bridge interactions are analyzed, including the inhibitor design targeting neuraminidase (NA) enzyme of influenza A virus, and the ligand binding interactions in the HCV p7 ion channel. The inhibition mechanism of the M2 proton channel in the influenza A virus is analyzed based on strong amino acid interactions.

Conclusion

(1) The salt bridge interactions between acidic amino acids (Glu^- and Asp^-) and alkaline amino acids (Arg⁺, Lys⁺ and His⁺) are the strongest residue-residue interactions. However, this type of interaction may be weakened by solvation effects and broken by lower pH conditions. (2) The cation- interactions between protonated amino acids (Arg⁺, Lys⁺ and His⁺) and aromatic amino acids (Phe, Tyr, Trp and His) are 2.5 to 5-fold stronger than common hydrogen bond interactions and are less affected by the solvation environment. (3) The amide bridge interactions between the two amide-containing amino acids (Asn and Gln) are three times stronger than hydrogen bond interactions, which are less influenced by the pH of the solution. (4) Ten of the twenty natural amino acids are involved in salt bridge, or cation-, or amide bridge interactions that often play important roles in protein-protein, protein-peptide, protein-ligand, and protein-DNA interactions.     

Contribution of a putative salt bridge and backbone dynamics in the structural instability of human prion protein upon R208H mutation

Molecular dynamics simulation method is used to assess the contribution of a disease-associated salt bridge in the early stages of the conformational rearrangement of human prion protein upon Arg²⁰⁸→His mutation, which causes Creutzfeldt-Jakob disease. Previous investigations have suggested that the breakage of this putative salt bridge (D¹⁴⁴/E¹⁴⁶ ↔ Arg²⁰⁸) between helix 1 and helix 3 is responsible for such a mutation-driven process. So far, no experimental data has been reported in order to distinguish the contribution of this single salt bridge in the initial steps of amyloid formation. Consequently, we decided to investigate the role of this salt bridge in early conformational rearrangements. To remove the salt bridge without perturbations in the backbone structure, the neutralized states of the involved residues were used. Three 10-ns molecular dynamics simulations on three initial structures have been performed. The results revealed that the early stages of the conformational rearrangements, against common belief, are mainly associated with the mutation-induced global changes in the backbone dynamics but not with the breaking of the salt bridge.     

Visual insight into how low pH alone can induce actin-severing ability in gelsolin under calcium-free conditions

Gelsolin is a key actin cytoskeleton-modulating protein primarily regulated by calcium and phosphoinositides. In addition, low pH has also been suggested to activate gelsolin in the absence of Ca²⁺ ions, although no structural insight on this pathway is available except for a reported decrement in its diffusion coefficient at low pH. We also observed ∼1.6-fold decrease in the molecular mobility of recombinant gelsolin when buffer pH was lowered from 9 to 5. Analysis of the small angle x-ray scattering data collected over the same pH range indicated that the radius of gyration and maximum linear dimension of gelsolin molecules increased from 30.3 to 34.1 Å and from 100 to 125 Å, respectively. Models generated for each dataset indicated that similar to the Ca²⁺-induced process, low pH also promotes unwinding of this six-domain protein but only partially. It appeared that pH is able to induce extension of the G1 domain from the rest of the five domains, whereas the Ca²⁺-sensitive latch between G2 and G6 domains remains closed. Interestingly, increasing the free Ca²⁺ level to merely ∼40 nm, the partially open pH 5 shape “sprung open” to a shape seen earlier for this protein at pH 8 and 1 mm free Ca²⁺. Also, pH alone could induce a shape where the g3-g4 linker of gelsolin was open when we truncated the C-tail latch from this protein. Our results provide insight into how under physiological conditions, a drop in pH can fully activate the F-actin-severing shape of gelsolin with micromolar levels of Ca²⁺ available.     

Structure and Mechanism of Human UDP-xylose Synthase: EVIDENCE FOR A PROMOTING ROLE OF SUGAR RING DISTORTION IN A THREE-STEP CATALYTIC CONVERSION OF UDP-GLUCURONIC ACID

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-d-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD⁺ and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-d-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the d-glucuronyl ring accommodated by UXS features a marked ⁴C₁ chair to B_O,3 boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C₄ (step 1) by aligning the enzymatic base Tyr¹⁴⁷ with the reactive substrate hydroxyl and it brings the carboxylate group at C₅ into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-d-glucuronic acid in the second chemical step. The protonated side chain of Tyr¹⁴⁷ stabilizes the enolate of decarboxylated C₄ keto species (²H₁ half-chair) that is then protonated from the Si face at C₅, involving water coordinated by Glu¹²⁰. Arg²⁷⁷, which is positioned by a salt-link interaction with Glu¹²⁰, closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C₄ keto group by NADH, assisted by Tyr¹⁴⁷ as catalytic proton donor, yields UDP-xylose adopting the relaxed ⁴C₁ chair conformation (step 3).     

A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O² of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and H²O². Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr¹⁶⁹ forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O·sugar complex. Transient kinetics of wild-type (WT) and Thr¹⁶⁹ → S/N/G/A replacement variants show that d-Glc binds to T169S, T169N, and WT with the same K_d (45–47 mm), and the hydride transfer rate constants (k^red) are similar (15.3–9.7 s⁻¹ at 4 °C). k^red of T169G with d-glucose (0.7 s⁻¹, 4 °C) is significantly less than that of WT but not as severely affected as in T169A (k^red of 0.03 s⁻¹ at 25 °C). Transient kinetics of WT and mutants using d-galactose show that P2O binds d-galactose with a one-step binding process, different from binding of d-glucose. In T169S, T169N, and T169G, the overall turnover with d-Gal is faster than that of WT due to an increase of k^red. In the crystal structure of T169S, Ser¹⁶⁹ Oγ assumes a position identical to that of Oγ1 in Thr¹⁶⁹; in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr¹⁶⁹ side chain are required for intermediate stabilization.     

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_cat/K_m = 1000 ± 100 m⁻¹ s⁻¹ versus 700 ± 100 m⁻¹ s⁻¹). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_cat/K_m = 80 ± 40 m⁻¹ s⁻¹). In the presence of 3-HSA the K_mapp for O₂ was 100 ± 10 μm. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme''s substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val³⁶⁷–Val³⁹⁴) could adopt two conformations differing by a rigid body rotation of 25° around Arg³⁶⁶. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme''s substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.