Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase

Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity.We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl₂ or MnCl₂. In the E122Q mutant, k_cat is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.     

Immunoglobulin G1 Fc Domain Motions: Implications for Fc Engineering

The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G (IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fcγ receptors (FcγRs) on immune cells. The cellular response and committal to a damaging, though protective, immune response are tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in FcγR affinity being modulated through shifts in Fc conformational sampling. Here, we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal Cγ2 domains and N-glycan than previously observed. Residues in the Cγ2/Cγ3 interface and disulfide-bonded hinge were identified as influencing the Cγ2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating FcγRs interconverted with Fc conformations distinct from those observed in FcγR complexes, which may represent a transient, nonbinding population.     

Crystal Structure of Prostate Secretory Protein PSP94 Shows an Edge-to-Edge Association of two Monomers to Form a Homodimer

Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins from prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis, and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with 10 conserved cysteine residues. PSP94 homologues show high-affinity binding with different proteins from the cysteine-rich secretory protein family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 Å resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last β-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion, leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to cysteine-rich secretory proteins and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface.     

Structural details of HIV-1 recognition by the broadly neutralizing monoclonal antibody 2F5: epitope conformation, antigen-recognition loop mobility, and anion-binding site

2F5 is a monoclonal antibody with potent and broadly neutralizing activity against HIV-1. It targets the membrane-proximal external region (MPER) of the gp41 subunit of the envelope glycoprotein and interferes with the process of fusion between viral and host cell membranes. This study presents eight 2F5 F_ab′ crystal structures in complex with various gp41 peptide epitopes. These structures reveal several key features of this antibody-antigen interaction. (1) Whenever free of contacts caused by crystal artifacts, the extended complementarity-determining region H3 loop is mobile; this is true for ligand-free and epitope-bound forms. (2) The interaction between the antibody and the gp41 ELDKWA epitope core is absolutely critical, and there are also close and specific contacts with residues located N-terminal to the epitope core. (3) Residues located at the C-terminus of the gp41 ELDKWA core do not interact as tightly with the antibody. However, in the presence of a larger peptide containing the gp41 fusion peptide segment, these residues adopt a conformation consistent with the start of an α-helix. (4) At high sulfate concentrations, the electron density maps of 2F5 F_ab′-peptide complexes contain a peak that may mark a binding site for phosphate groups of negatively charged lipid headgroups. The refined atomic-level details of 2F5 paratope-epitope interactions revealed here should contribute to a better understanding of the mechanism of 2F5-based virus neutralization, in general, and prove important for the design of potential vaccine candidates intended to elicit 2F5-like antibody production.     

Structures of Intermediates along the Catalytic Cycle of Terminal Deoxynucleotidyltransferase: Dynamical Aspects of the Two-Metal Ion Mechanism

Terminal deoxynucleotidyltransferase (Tdt) is a non-templated eukaryotic DNA polymerase of the polX family that is responsible for the random addition of nucleotides at the V(D)J junctions of immunoglobulins and T-cell receptors. Here we describe a series of high-resolution X-ray structures that mimic the pre-catalytic state, the post-catalytic state and a competent state that can be transformed into the two other ones in crystallo via the addition of dAMPcPP and Zn^2 +, respectively. We examined the effect of Mn^2 +, Co^2 + and Zn^2 + because they all have a marked influence on the kinetics of the reaction. We demonstrate a dynamic role of divalent transition metal ions bound to site A: (i) Zn^2 + (or Co^2 +) in Metal A site changes coordination from octahedral to tetrahedral after the chemical step, which explains the known higher affinity of Tdt for the primer strand when these ions are present, and (ii) metal A has to leave to allow the translocation of the primer strand and to clear the active site, a typical feature for a ratchet-like mechanism. Except for Zn^2 +, the sugar puckering of the primer strand 3′ terminus changes from C2′-endo to C3′-endo during catalysis. In addition, our data are compatible with a scheme where metal A is the last component that binds to the active site to complete its productive assembly, as already inferred in human pol beta. The new structures have potential implications for modeling pol mu, a closely related polX implicated in the repair of DNA double-strand breaks, in a complex with a DNA synapsis.     

Role of bundle helices in a regulatory crosstalk in the trimeric betaine transporter BetP

The Na⁺-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na⁺-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.     

The role of multiple hydrogen-bonding groups in specific alcohol binding sites in proteins: insights from structural studies of LUSH

It is now generally accepted that many of the physiological effects of alcohol consumption are a direct result of binding to specific sites in neuronal proteins such as ion channels or other components of neuronal signaling cascades. Binding to these targets generally occurs in water-filled pockets and leads to alterations in protein structure and dynamics. However, the precise interactions required to confer alcohol sensitivity to a particular protein remain undefined.Using information from the previously solved crystal structures of the Drosophila melanogaster protein LUSH in complexes with short-chain alcohols, we have designed and tested the effects of specific amino acid substitutions on alcohol binding. The effects of these substitutions, specifically S52A, T57S, and T57A, were examined using a combination of molecular dynamics, X-ray crystallography, fluorescence spectroscopy, and thermal unfolding. These studies reveal that the binding of ethanol is highly sensitive to small changes in the composition of the alcohol binding site. We find that T57 is the most critical residue for binding alcohols; the T57A substitution completely abolishes binding, while the T57S substitution differentially affects ethanol binding compared to longer-chain alcohols. The additional requirement for a potential hydrogen-bond acceptor at position 52 suggests that both the presence of multiple hydrogen-bonding groups and the identity of the hydrogen-bonding residues are critical for defining an ethanol binding site. These results provide new insights into the detailed chemistry of alcohol's interactions with proteins.     

Disulfide Bond Stabilization of the Hexameric Capsomer of Human Immunodeficiency Virus

The human immunodeficiency virus type 1 capsid is modeled as a fullerene cone that is composed of ∼ 250 hexamers and 12 pentamers of the viral CA protein. Structures of CA hexamers have been difficult to obtain because the hexamer-stabilizing interactions are inherently weak, and CA tends to spontaneously assemble into capsid-like particles. Here, we describe a two-step biochemical strategy to obtain soluble CA hexamers for crystallization. First, the hexamer was stabilized by engineering disulfide cross-links (either A14C/E45C or A42C/T54C) between the N-terminal domains of adjacent subunits. Second, the cross-linked hexamers were prevented from polymerizing further into hyperstable capsid-like structures by mutations (W184A and M185A) that interfered with dimeric association between the C-terminal domains that link adjacent hexamers. The structures of two different cross-linked CA hexamers were nearly identical, and we combined the non-mutated portions of the structures to generate an atomic resolution model for the native hexamer. This hybrid approach for structure determination should be applicable to other viral capsomers and protein-protein complexes in general.     

Reduced phosphatase activity of SHP-2 in LEOPARD syndrome: consequences for PI3K binding on Gab1

LEOPARD (LS) and Noonan (NS) are overlapping syndromes associated with distinct mutations of SHP-2. Whereas NS mutations enhance SHP-2 catalytic activity, we show that the activity of three representative LS mutants is undetectable when assayed using a standard protein tyrosine phosphatase (PTP) substrate. A different assay using a specific SHP-2 substrate confirms their decreased PTP activity, but also reveals a significant activity of the T468M mutant. In transfected cells stimulated with epidermal growth factor, the least active LS mutants promote Gab1/PI3K binding, validating our in vitro data. LS mutants thus display a reduced PTP activity both in vitro and in transfected cells.     

The Crystal Structure of the Pseudomonas dacunhae Aspartate-β-Decarboxylase Dodecamer Reveals an Unknown Oligomeric Assembly for a Pyridoxal-5′-Phosphate-Dependent Enzyme

The Pseudomonas dacunhael-aspartate-β-decarboxylase (ABDC, aspartate 4-decarboxylase, aspartate 4-carboxylyase, E.C. 4.1.1.12) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-decarboxylation of l-aspartate to produce l-alanine and CO₂. This catalytically versatile enzyme is known to form functional dodecamers at its optimal pH and is thought to work in conjunction with an l-Asp/l-Ala antiporter to establish a proton gradient across the membrane that can be used for ATP biosynthesis. We have solved the atomic structure of ABDC to 2.35 Å resolution using single-wavelength anomalous dispersion phasing. The structure reveals that ABDC oligomerizes as a homododecamer in an unknown mode among PLP-dependent enzymes and has highest structural homology with members of the PLP-dependent aspartate aminotransferase subfamily. The structure shows that the ABDC active site is very similar to that of aspartate aminotransferase. However, an additional arginine side chain (Arg37) was observed flanking the re-side of the PLP ring in the ABDC active site. The mutagenesis results show that although Arg37 is not required for activity, it appears to be involved in the ABDC catalytic cycle.     

Glutamate versus glutamine exchange swaps substrate selectivity in tRNA-guanine transglycosylase: insight into the regulation of substrate selectivity by kinetic and crystallographic studies

Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of guanine in the wobble position of particular tRNAs by the modified base preQ₁. In vitro, however, the enzyme is also able to insert the immediate biosynthetic precursor, preQ₀, into those tRNAs. This substrate promiscuity is based on a peptide switch in the active site, gated by the general acid/base Glu235. The switch alters the properties of the binding pocket to allow either the accommodation of guanine or preQ₁. The peptide conformer recognising guanine, however, is also able to bind preQ₀. To investigate selectivity regulation, kinetic data for Zymomonas mobilis Tgt were recorded. They show that selectivity in favour of the actual substrate preQ₁ over preQ₀ is not achieved by a difference in affinity but via a higher turnover rate. Moreover, a Tgt(Glu235Gln) variant was constructed. The mutation was intended to stabilise the peptide switch in the conformation favouring guanine and preQ₀ binding. Kinetic characterisation of the mutated enzyme revealed that the Glu235Gln exchange has, with respect to all substrate bases, no significant influence on k_cat. In contrast, K_M(preQ₁) is drastically increased, while K_M(preQ₀) seems to be decreased. Hence, regarding k_cat/K_M as an indicator for catalytic efficiency, selectivity of Tgt in favour of preQ₁ is abolished or even inverted in favour of preQ₀ for Tgt(Glu235Gln). Crystal structures of the mutated enzyme confirm that the mutation strongly favours the binding pocket conformation required for the accommodation of guanine and preQ₀. The way this is achieved, however, significantly differs from that predicted based on crystal structures of wild-type Tgt.     

Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor

Human neutrophil elastase (HNE), a trypsin-type serine protease, is of pivotal importance in the onset and progression of chronic obstructive pulmonary disease (COPD). COPD encompasses a group of slowly progressive respiratory disorders and is a major medical problem and the fifth leading cause of death worldwide. HNE is a major target for the development of compounds that inhibit the progression of long-term lung function decline in COPD patients.Here, we present the three-dimensional structure of a potent dihydropyrimidone inhibitor (DHPI) non-covalently bound to HNE at a resolution of 2.0 Å. The inhibitor binds to the active site in a unique orientation addressing S1 and S2 subsites of the protease. To facilitate further analysis of this binding mode, we determined the structure of the uncomplexed enzyme at a resolution of 1.86 Å. Detailed comparisons of the HNE:DHPI complex with the uncomplexed HNE structure and published structures of other elastase:inhibitor complexes revealed that binding of DHPI leads to large conformational changes in residues located in the S2 subsite. The rearrangement of residues Asp95-Leu99B creates a deep, well-defined cavity, which is filled by the P2 moiety of the inhibitor molecule to almost perfect shape complementarity. The shape of the S2 subsite in complex with DHPI clearly differs from all other observed HNE structures. The observed structural flexibility of the S2 subsite is a key feature for the understanding of the binding mode of DHPIs in general and the development of new HNE selective inhibitors.     

Conserved glycine residues in the P-loop of ATP synthases form a doorframe for nucleotide entrance

The phosphate binding loop (GXXXXGKT(S)) is conserved in several mononucleotide-binding proteins with similar three-dimensional structures. Although variations in other amino acids have been noted, the first glycine and glycine-lysine residues are highly conserved in all enzymes, whose role is yet to be understood. Alanine substitutions for critically positioned glycines—G234, G237, and G239—were generated for the catalytic A-subunit of A-ATP synthase from Pyrococcus horikoshii OT3, and their crystal structures were determined. They showed altered conformation for the phosphate binding loop, with G234A and G237A becoming flat and with G239A taking an intermediate conformation, resulting in the active-site region being closed to nucleotide entry. Furthermore, the essential amino acids S238 and K240, which normally interact with the nucleotide, become inaccessible. These mutant structures demonstrate the role of the strictly conserved glycine residues in guarding the active-site region for nucleotide entrance in archaea-type ATP synthases.     

A crystallographic study of the role of sequence context in thymine glycol bypass by a replicative DNA polymerase serendipitously sheds light on the exonuclease complex

Thymine glycol (Tg) is the most common oxidation product of thymine and is known to be a strong block to replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5′-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized thymine displaces the adjacent 5′-G, which can no longer serve as a template for primer elongation [Aller, P., Rould, M. A., Hogg, M, Wallace, S. S. & Doublié S. (2007). A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol. Proc. Natl. Acad. Sci. USA, 104, 814-818.].Several studies showed that in the sequence context 5′-C-Tg-purine, Tg is more likely to be bypassed by Klenow fragment, an A-family DNA polymerase. We set out to investigate the role of sequence context in Tg bypass in a B-family polymerase and to solve the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg-containing DNA in the three remaining sequence contexts: 5′-A-Tg-G, 5′-T-Tg-G, and 5′-C-Tg-G. A combination of several factors—including the associated exonuclease activity, the nature of the 3′ and 5′ bases surrounding Tg, and the cis-trans interconversion of Tg—influences Tg bypass. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and in the stabilization of the primer strand in the exonuclease site.     

Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5′-TTTGTTCTCTAAAGAGAACTTA-3′, containing a pair of SinR consensus sequences in inverted orientation with a K_d of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which “repurposes” SinR as a repressor of autolysin and motility genes, are discussed.     

Structural and thermodynamic comparison of the catalytic domain of AMSH and AMSH-LP: nearly identical fold but different stability

AMSH plays a critical role in the ESCRT (endosomal sorting complexes required for transport) machinery, which facilitates the down-regulation and degradation of cell-surface receptors. It displays a high level of specificity toward cleavage of Lys63-linked polyubiquitin chains, the structural basis of which has been understood recently through the crystal structure of a highly related, but ESCRT-independent, protein AMSH-LP (AMSH-like protein). We have determined the X-ray structure of two constructs representing the catalytic domain of AMSH: AMSH244, the JAMM (JAB1/MPN/MOV34)-domain-containing polypeptide segment from residues 244 to 424, and AMSH219^E280A, an active-site mutant, Glu280 to Ala, of the segment from 219 to 424. In addition to confirming the expected zinc coordination in the protein, the structures reveal that the catalytic domains of AMSH and AMSH-LP are nearly identical; however, guanidine-hydrochloride-induced unfolding studies show that the catalytic domain of AMSH is thermodynamically less stable than that of AMSH-LP, indicating that the former is perhaps structurally more plastic. Much to our surprise, in the AMSH219^E280A structure, the catalytic zinc was still held in place, by the compensatory effect of an aspartate from a nearby loop moving into a position where it could coordinate with the zinc, once again suggesting the plasticity of AMSH. Additionally, a model of AMSH244 bound to Lys63-linked diubiquitin reveals a type of interface for the distal ubiquitin significantly different from that seen in AMSH-LP. Altogether, we believe that our data provide important insight into the structural difference between the two proteins that may translate into the difference in their biological function.