Ribonucleotide reductase (RR) is an αnβn (RR1●RR2) complex that maintains balanced dNTP pools by reducing ribonucleoside diphosphates to deoxyribonucleoside diphosphates. RR1 is the catalytic subunit and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP-only, dATP-only, TTP●GDP, TTP●ATP, and TTP●dATP. These structures provide insights into ATP/dATP regulation of RR. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1●dATP hexamer and used single-particle electron microscopy to visualize the α6●ββ’ 1●dATP holo complex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data provide an elegant mechanism for regulating RR activity by dATP-induced oligomerization.

The Toll/interleukin-1 receptor (TIR) domain is a highly conserved signaling domain found in the intracellular regions of Toll-like receptors (TLRs), in interleukin-1 receptors, and in several cytoplasmic adaptor proteins. TIR domains mediate receptor signal transduction through recruitment of adaptor proteins and play critical roles in the innate immune response and inflammation. This work presents the 2.2 Å crystal structure of the TIR domain of human TLR10, revealing a symmetric dimer in the asymmetric unit. The dimer interaction surface contains residues from the BB-loop, DD-loop, and ␣C-helix, which have previously been identified as important structural motifs for signaling in homologous TLR receptors. The interaction surface is extensive, containing a central hydrophobic patch surrounded by polar residues. The BB-loop forms a tight interaction, where a range of consecutive residues binds in a pocket formed by the reciprocal BB-loop and ␣C-helix. This pocket appears to be well suited for binding peptide substrates, which is consistent with the notion that peptides and peptide mimetics of the BB-loop are inhibitors for TLR signaling. The TLR10 structure is in good agreement with available biochemical data on TLR receptors and is likely to provide a good model for the physiological dimer.The Toll-like receptors (TLRs) 3 are type I transmembrane receptors (1) that play an important role in innate immunity as they recognize conserved pathogen signatures and induce early gene expression responses to infections (2, 3). TLR signaling is mediated through the cytoplasmic Toll/interleukin-1 receptor (TIR) domain, the oligomerization of which initiates the recruitment of TIR domain-containing adaptor proteins. The cytoplasmic adaptor subgroup comprises five members: MyD88, MyD88-adaptor-like (MAL), TIR-domain-containing adaptor protein inducing IFNbeta (TRIF), TRIF-related adaptor molecule (TRAM), and sterile ␣-and armadillo-motif containing protein (SARM) (4), which mediate or modulate the downstream signaling. Subsequent kinase activation results in the induction of transcription factors such as interferon regulatory factor and NF-B followed by the production of numerous regulators and effectors of the innate immune response. In addition to the TLRs and the cytoplasmic TIR adaptor subgroup, the family of TIR domain-containing proteins encompasses the interleukin-1 (IL-1) receptor family (5).The 10 members of the human TLR family recognize a wide variety of pathogen-associated molecular patterns. As no ligand has yet been reported for TLR10, it remains the only orphan family member. Activation of TLRs is believed to involve receptor oligomerization, and the current view is that the receptors are active as either homodimers or heterodimers, depending on the receptor type. The cytoplasmic TIR domains dimerize to form a platform for the recruitment of adaptor proteins and additional signaling molecules. TLR10 has been shown to form homodimers and to interact with TLRs 1 and 2 (6). All of the TLRs, except TLR3, have b...

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Axonal growth cone guidance is a central process in nervous system development and repair. Collapsin response mediator protein 2 (CRMP-2) is a neurite extension-promoting neuronal cytosolic molecule involved in the signalling of growth inhibitory cues from external stimuli, such as semaphorin 3A and the myelin-associated glycoprotein. We have determined the crystal structure of human tetrameric CRMP-2, which is structurally related to the dihydropyriminidases; however, the active site is not conserved. The wealth of earlier functional mapping data for CRMP-2 are discussed in light of the threedimensional structure of the protein. The differences in oligomerisation interfaces between CRMP-1 and CRMP-2 are used to model CRMP-1/2 heterotetramers.

IMP dehydrogenase (IMPDH) and GMP reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH/GMPR family have not been identified. Here, we show that the GMPR reaction utilizes the same intermediate E-XMP* as IMPDH, but this intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimic a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations, an "in" conformation poised for hydride transfer, and an "out" conformation where the cofactor is 6 Å from IMP. Mutagenesis, substrate/cofactor analog experiments demonstrate that the “out” conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery activating ammonia.

Evasion of apoptosis is recognized as a characteristic of malignant growth. Anti-apoptotic B-cell lymphoma-2 (Bcl-2) family members have therefore emerged as potential therapeutic targets due to their critical role in proliferating cancer cells. Here, we present the crystal structure of Bfl-1, the last antiapoptotic Bcl-2 family member to be structurally characterized, in complex with a peptide corresponding to the BH3 region of the pro-apoptotic protein Bim. The structure reveals distinct features at the peptide-binding site, likely to define the binding specificity for pro-apoptotic proteins. Superposition of the Bfl-1:Bim complex with that of Mcl-1:Bim reveals a significant local plasticity of hydrophobic interactions contributed by the Bim peptide, likely to be the basis for the multi specificity of Bim for antiapoptotic proteins.

SHIP2, OCRL, and INPP5B belong to inositol polyphosphate 5-phophatase subfamilies involved in insulin regulation and Lowes syndrome. The structural basis for membrane recognition, substrate specificity, and regulation of inositol polyphosphate 5-phophatases is still poorly understood. We determined the crystal structures of human SHIP2, OCRL, and INPP5B, the latter in complex with phosphoinositide substrate analogs, which revealed a membrane interaction patch likely to assist in sequestering substrates from the lipid bilayer. Residues recognizing the 1-phosphate of the substrates are highly conserved among human family members, suggesting similar substrate binding modes. However, 3- and 4-phosphate recognition varies and determines individual substrate specificity profiles. The high conservation of the environment of the scissile 5-phosphate suggests a common reaction geometry for all members of the human 5-phosphatase family.

Guanine monophosphate (GMP) synthetase is a bifunctional two-domain enzyme. The N-terminal glutaminase domain generates ammonia from glutamine and the C-terminal synthetase domain aminates xanthine monophosphate (XMP) to form GMP. Mammalian GMP synthetases (GMPSs) contain a 130-residue-long insert in the synthetase domain in comparison to bacterial proteins. We report here the structure of a eukaryotic GMPS. Substrate XMP was bound in the crystal structure of the human GMPS enzyme. XMP is bound to the synthetase domain and covered by a LID motif. The enzyme forms a dimer in the crystal structure with subunit orientations entirely different from the bacterial counterparts. The inserted sub-domain is shown to be involved in substrate binding and dimerization. Furthermore, the structural basis for XMP recognition is revealed as well as a potential allosteric site. Enzymes in the nucleotide metabolism typically display an increased activity in proliferating cells due to the increased need for nucleotides. Many drugs used as immunosuppressants and for treatment of cancer and viral diseases are indeed nucleobase- and nucleoside-based compounds, which are acting on or are activated by enzymes in this pathway. The information obtained from the crystal structure of human GMPS might therefore aid in understanding interactions of nucleoside-based drugs with GMPS and in structure-based design of GMPS-specific inhibitors.