Temperature-dependent iron motion in extremophile rubredoxins - no need for 'corresponding states'.

Extremophile organisms are known that can metabolize at temperatures down to - 25 °C (psychrophiles) and up to 122 °C (hyperthermophiles). Understanding viability under extreme conditions is relevant for human health, biotechnological applications, and our search for life elsewhere in the universe. Information about the stability and dynamics of proteins under environmental extremes is an important factor in this regard. Here we compare the dynamics of small Fe-S proteins - rubredoxins - from psychrophilic and hyperthermophilic microorganisms, using three different nuclear techniques as well as molecular dynamics calculations to quantify motion at the Fe site. The theory of 'corresponding states' posits that homologous proteins from different extremophiles have comparable flexibilities at the optimum growth temperatures of their respective organisms. Although 'corresponding states' would predict greater flexibility for rubredoxins that operate at low temperatures, we find that from 4 to 300 K, the dynamics of the Fe sites in these homologous proteins are essentially equivalent.

Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1 ubiquitin-associated (UBA) domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. No experimentally determined molecular structure of this unusual UBA domain is available. We fused the UBA domain to the 1TEL variant of the translocation ETS leukemia protein sterile alpha motif (TELSAM) crystallization chaperone and obtained crystals diffracting as far as 1.53 Å. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and crystallize at protein concentrations as low as 0.2 mg/mL. Our studies support a mechanism of 1TEL fusion crystallization and show that 1TEL fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1-UBA domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. Sequence analysis suggests an unusual architecture for the TNK1 UBA domain, but an experimentally-validated molecular structure is undetermined. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 Å. A 1TEL search model enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our studies support a mechanism of TELSAM fusion crystallization and show that TELSAM fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

Structure of human cytomegalovirus UL144, an HVEM orthologue, bound to the B and T cell lymphocyte attenuator.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that has co-evolved with the host immune system to establish lifelong persistence. HCMV encodes many immunomodulatory molecules, including the glycoprotein UL144. UL144 is a structural mimic of the tumor necrosis factor receptor superfamily member HVEM (herpesvirus entry mediator), which binds to the various ligands LIGHT, LTα, BTLA, CD160, and gD. However, in contrast to HVEM, UL144 only binds BTLA, inhibiting T-cell activation. Here, we report the crystal structure of the UL144-BTLA complex, revealing that UL144 utilizes residues from its N-terminal cysteine-rich domain 1 (CRD1) to interact uniquely with BTLA. The shorter CRD2 loop of UL144 also alters the relative orientation of BTLA binding with both N-terminal CRDs. By employing structure-guided mutagenesis, we have identified a mutant of BTLA (L123A) that interferes with HVEM binding but preserves UL144 interactions. Furthermore, our results illuminate structural differences between UL144 and HVEM that explain its binding selectivity and highlight it as a suitable scaffold for designing superior, immune inhibitory BTLA agonists.

Crystal structure of the m4-1BB/4-1BBL complex reveals an unusual dimeric ligand that undergoes structural changes upon 4-1BB receptor binding.

The interaction between the receptor 4-1BB and its ligand 4-1BBL provides co-stimulatory signals for T-cell activation and proliferation. However, differences in the mouse and human molecules might result in differential engagement of this pathway. Here, we report the crystal structure of mouse 4-1BBL and of the mouse 4-1BB/4-1BBL complex, which together provided insights into the molecular mechanism by which m4-1BBL and its cognate receptor recognize each other. Unlike all human or mouse tumor necrosis factor ligands that form noncovalent and mostly trimeric assemblies, the m4-1BBL structure formed a disulfide-linked dimeric assembly. The structure disclosed that certain differences in the amino acid composition along the intramolecular interface, together with two specific residues (Cys-246 and Ser-256) present exclusively in m4-1BBL, are responsible for this unique dimerization. Unexpectedly, upon m4-1BB binding, m4-1BBL undergoes structural changes within each protomer; moreover, the individual m4-1BBL protomers rotate relative to each other, yielding a dimerization interface with more inter-subunit interactions. We also observed that in the m4-1BB/4-1BBL complex, each receptor monomer binds exclusively to a single ligand subunit with contributions of cysteine-rich domain 1 (CRD1), CRD2, and CRD3. Furthermore, structure-guided mutagenesis of the binding interface revealed that novel binding interactions with the GH loop, rather than the DE loop, are energetically critical and define the m4-1BB receptor selectivity for m4-1BBL. A comparison with the human 4-1BB/4-1BBL complex highlighted several differences between the ligand- and receptor-binding interfaces, providing an explanation for the absence of inter-species cross-reactivity between human and mouse 4-1BB and 4-1BBL molecules.

Crystal structures of the human 4-1BB receptor bound to its ligand 4-1BBL reveal covalent receptor dimerization as a potential signaling amplifier.

Human (h)4-1BB (TNFRSF9 or CD137) is an inducible tumor necrosis factor receptor (TNFR) superfamily member that interacts with its cognate ligand h4-1BBL to promote T lymphocyte activation and proliferation. h4-1BB is currently being targeted with agonists in cancer immunotherapy. Here, we determined the crystal structures of unbound h4-1BBL and both WT h4-1BB and a dimerization-deficient h4-1BB mutant (C121S) in complex with h4-1BBL at resolutions between 2.7 and 3.2 Å. We observed that the structural arrangement of 4-1BBL, both unbound and in the complex, represents the canonical bell shape as seen in other similar TNF proteins and differs from the previously reported three-bladed propeller structure of 4-1BBL. We also found that the binding site for the receptor is at the crevice formed between two protomers of h4-1BBL, but that h4-1BB interacts predominantly with only one ligand protomer. Moreover, h4-1BBL lacked the conserved tyrosine residue in the DE loop that forms canonical interactions between other TNFR family molecules and their ligands, suggesting h4-1BBL engages h4-1BB through a distinct mechanism. Of note, we discovered that h4-1BB forms a disulfide-linked dimer because of the presence of an additional cysteine residue found in its cysteine-rich domain 4 (CRD4). As a result, h4-1BB dimerization, in addition to trimerization via h4-1BBL binding, could result in cross-linking of individual ligand-receptor complexes to form a 2D network that stimulates strong h4-1BB signaling. This work provides critical insights into the structural and functional properties of both h4-1BB and h4-1BBL and reveals that covalent receptor dimerization amplifies h4-1BB signaling.

Structure-function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8.

Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, and VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138- and VACV-304-binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.

Crystal structure of murine 4-1BB and its interaction with 4-1BBL support a role for galectin-9 in 4-1BB signaling.

4-1BB (CD137) is a TNF receptor superfamily (TNFRSF) member that is thought to undergo receptor trimerization upon binding to its trimeric TNF superfamily ligand (4-1BBL) to stimulate immune responses. 4-1BB also can bind to the tandem repeat-type lectin galectin-9 (Gal-9), and signaling through mouse (m)4-1BB is reduced in galectin-9 (Gal-9)-deficient mice, suggesting a pivotal role of Gal-9 in m4-1BB activation. Here, using sulfur-SAD phasing, we determined the crystal structure of m4-1BB to 2.2-Å resolution. We found that similar to other TNFRSFs, m4-1BB has four cysteine-rich domains (CRDs). However, the organization of CRD1 and the orientation of CRD3 and CRD4 with respect to CRD2 in the m4-1BB structure distinctly differed from those of other TNFRSFs. Moreover, we mapped two Asn residues within CRD4 that are N-linked glycosylated and mediate m4-1BB binding to Gal-9. Kinetics studies of m4-1BB disclosed a very tight nanomolar binding affinity to m4-1BBL with an unexpectedly strong avidity effect. Both N- and C-terminal domains of Gal-9 bound m4-1BB, but with lower affinity compared with m4-1BBL. Although the TNF homology domain (THD) of human (h)4-1BBL forms non-covalent trimers, we found that m4-1BBL formed a covalent dimer via 2 cysteines absent in h4-1BBL. As multimerization and clustering is a prerequisite for TNFR intracellular signaling, and as m4-1BBL can only recruit two m4-1BB monomers, we hypothesize that m4-1BBL and Gal-9 act together to aid aggregation of m4-1BB monomers to efficiently initiate m4-1BB signaling.

Structural and functional analysis of the human POT1-TPP1 telomeric complex.

POT1 and TPP1 are part of the shelterin complex and are essential for telomere length regulation and maintenance. Naturally occurring mutations of the telomeric POT1-TPP1 complex are implicated in familial glioma, melanoma and chronic lymphocytic leukaemia. Here we report the atomic structure of the interacting portion of the human telomeric POT1-TPP1 complex and suggest how several of these mutations contribute to malignant cancer. The POT1 C-terminus (POT1C) forms a bilobal structure consisting of an OB-fold and a holiday junction resolvase domain. TPP1 consists of several loops and helices involved in extensive interactions with POT1C. Biochemical data shows that several of the cancer-associated mutations, partially disrupt the POT1-TPP1 complex, which affects its ability to bind telomeric DNA efficiently. A defective POT1-TPP1 complex leads to longer and fragile telomeres, which in turn promotes genomic instability and cancer.

Structure and Function of a Novel ATPase that Interacts with Holliday Junction Resolvase Hjc and Promotes Branch Migration.

Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicusPilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage.

Crystal structure of the Leishmania major peroxidase-cytochrome c complex.

The causative agent of leishmaniasis is the protozoan parasite Leishmania major. Part of the host protective mechanism is the production of reactive oxygen species including hydrogen peroxide. In response, L. major produces a peroxidase, L. major peroxidase (LmP), that helps to protect the parasite from oxidative stress. LmP is a heme peroxidase that catalyzes the peroxidation of mitochondrial cytochrome c. We have determined the crystal structure of LmP in a complex with its substrate, L. major cytochrome c (LmCytc) to 1.84 Å, and compared the structure to its close homolog, the yeast cytochrome c peroxidase-cytochrome c complex. The binding interface between LmP and LmCytc has one strong and one weak ionic interaction that the yeast system lacks. The differences between the steady-state kinetics correlate well with the Lm redox pair being more dependent on ionic interactions, whereas the yeast redox pair depends more on nonpolar interactions. Mutagenesis studies confirm that the ion pairs at the intermolecular interface are important to both k(cat) and K(M). Despite these differences, the electron transfer path, with respect to the distance between hemes, along the polypeptide chain is exactly the same in both redox systems. A potentially important difference, however, is the side chains involved. LmP has more polar groups (Asp and His) along the pathway compared with the nonpolar groups (Leu and Ala) in the yeast system, and as a result, the electrostatic environment along the presumed electron transfer path is substantially different.

Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD+ synthetase.

Glutamine-dependent NAD+ synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD+ from NaAD+ (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 Å (1 Å=0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligand complexes with substrates (NaAD+/ATP), substrate analogue {NaAD+/AMP-CPP (adenosine 5'-[α,ß-methylene]triphosphate)} and intermediate analogues (NaAD+/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD+/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

Structure and function of P19, a high-affinity iron transporter of the human pathogen Campylobacter jejuni.

Campylobacter jejuni, a major cause of acute bacterial diarrhea in humans, expresses numerous proteins to import diverse forms of essential iron. The expression of p19 and an adjacent iron transporter homologue (ftr1) is strongly induced upon iron limitation, suggesting a function in iron acquisition. Here, we show that the loss of P19 alone is detrimental to growth on iron-restricted media. Furthermore, metal binding analysis demonstrates that recombinant P19 has distinct copper and iron binding sites. Crystal structures of P19 have been solved to 1.41 A resolution, revealing an immunoglobulin-like fold. A P19 homodimer in which both monomers contribute ligands to two equivalent copper sites located adjacent to methionine-rich patches is observed. Copper coordination occurs via three histidine residues (His42, His95, and His132) and Met88. A solvent channel lined with conserved acidic residues leads to the copper site. Soaking crystals with a solution of manganese as iron analog reveals a second metal binding site in this solvent channel (metal-metal distance, 7.7 A). Glu44 lies between the metal sites and displays multiple conformations in the crystal structures, suggesting a role in regulating metal-metal interaction. Dimerization is shown to be metal dependent in vitro and is detected in vivo by cross-linking.

Heme-coordinating inhibitors of neuronal nitric oxide synthase. Iron-thioether coordination is stabilized by hydrophobic contacts without increased inhibitor potency.

The heme-thioether ligand interaction often occurs between heme iron and native methionine ligands, but thioether-based heme-coordinating (type II) inhibitors are uncommon due to the difficulty in stabilizing the Fe-S bond. Here, a thioether-based inhibitor (3) of neuronal nitric oxide synthase (nNOS) was designed, and its binding was characterized by spectrophotometry and crystallography. A crystal structure of inhibitor 3 coordinated to heme iron was obtained, representing, to our knowledge, the first crystal structure of a thioether inhibitor complexed to any heme enzyme. A series of related potential inhibitors (4-8) also were evaluated. Compounds 4-8 were all found to be type I (non-heme-coordinating) inhibitors of ferric nNOS, but 4 and 6-8 were found to switch to type II upon heme reduction to the ferrous state, reflecting the higher affinity of thioethers for ferrous heme than for ferric heme. Contrary to what has been widely thought, thioether-heme ligation was found not to increase inhibitor potency, illustrating the intrinsic weakness of the thioether-ferric heme linkage. Subtle changes in the alkyl groups attached to the thioether sulfur caused drastic changes in the binding conformation, indicating that hydrophobic contacts play a crucial role in stabilizing the thioether-heme coordination.

Combined microspectrophotometric and crystallographic examination of chemically reduced and X-ray radiation-reduced forms of cytochrome ba3 oxidase from Thermus thermophilus: structure of the reduced form of the enzyme.

Three paths for obtaining crystals of reduced (II-E4Q/I-K258R) cytochrome ba(3) are described, and the structures of these are reported at approximately 2.8-3.0 A resolution. Microspectrophotometry of single crystals of Thermus ba(3) oxidase at 100 K was used to show that crystals of the oxidized enzyme are reduced in an intense X-ray (beam line 7-1 at the Stanford Synchrotron Radiation Laboratory), being nearly complete in 1 min. The previously reported structures of ba(3) (Protein Data Bank entries 1EHK and 1XME ), having a crystallographically detectable water between the Cu(B) and Fe(a3) metals of the dinuclear center, actually represent the X-ray radiation-reduced enzyme. Dithionite-reduced crystals or crystals formed from dithionite-reduced enzyme revealed the absence of the above-mentioned water and an increase in the Cu(B)-Fe(a3) distance of approximately 0.3 A. The new structures are discussed in terms of enzyme function. An unexpected optical absorption envelope at approximately 590 nm is also reported. This spectral feature is tentatively thought to arise from a five-coordinate, low-spin, ferrous heme a(3) that is trapped in the frozen crystals.