Atomic structure of Lanreotide nanotubes revealed by cryo-EM.

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

BRCA2 binding through a cryptic repeated motif to HSF2BP oligomers does not impact meiotic recombination.

BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.

Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation.

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ's dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

The Multifaceted Roles of Ku70/80.

DNA double-strand breaks (DSBs) are accidental lesions generated by various endogenous or exogenous stresses. DSBs are also genetically programmed events during the V(D)J recombination process, meiosis, or other genome rearrangements, and they are intentionally generated to kill cancer during chemo- and radiotherapy. Most DSBs are processed in mammalian cells by the classical nonhomologous end-joining (c-NHEJ) pathway. Understanding the molecular basis of c-NHEJ has major outcomes in several fields, including radiobiology, cancer therapy, immune disease, and genome editing. The heterodimer Ku70/80 (Ku) is a central actor of the c-NHEJ as it rapidly recognizes broken DNA ends in the cell and protects them from nuclease activity. It subsequently recruits many c-NHEJ effectors, including nucleases, polymerases, and the DNA ligase 4 complex. Beyond its DNA repair function, Ku is also involved in several other DNA metabolism processes. Here, we review the structural and functional data on the DNA and RNA recognition properties of Ku implicated in DNA repair and in telomeres maintenance.

Design on a Rational Basis of High-Affinity Peptides Inhibiting the Histone Chaperone ASF1.

Anti-silencing function 1 (ASF1) is a conserved H3-H4 histone chaperone involved in histone dynamics during replication, transcription, and DNA repair. Overexpressed in proliferating tissues including many tumors, ASF1 has emerged as a promising therapeutic target. Here, we combine structural, computational, and biochemical approaches to design peptides that inhibit the ASF1-histone interaction. Starting from the structure of the human ASF1-histone complex, we developed a rational design strategy combining epitope tethering and optimization of interface contacts to identify a potent peptide inhibitor with a dissociation constant of 3 nM. When introduced into cultured cells, the inhibitors impair cell proliferation, perturb cell-cycle progression, and reduce cell migration and invasion in a manner commensurate with their affinity for ASF1. Finally, we find that direct injection of the most potent ASF1 peptide inhibitor in mouse allografts reduces tumor growth. Our results open new avenues to use ASF1 inhibitors as promising leads for cancer therapy.

A higher-order entity formed by the flexible assembly of RAP1 with TRF2.

Telomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shelterin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments.

One identity or more for telomeres?

A major issue in telomere research is to understand how the integrity of chromosome ends is controlled. The fact that different types of nucleoprotein complexes have been described at the telomeres of different organisms raises the question of whether they have in common a structural identity that explains their role in chromosome protection. We will review here how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA, and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guarantee the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We will also discuss the recent notion that telomeres have evolved specific systems to overcome the DNA topological stress generated during their replication and transcription. This will lead to revisit the way we envisage the functioning of telomeric complexes since the regulation of topology is central to DNA stability, replication, recombination, and transcription as well as to chromosome higher-order organization.

Structure of the MutLα C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site.

Mismatch-repair factors have a prominent role in surveying eukaryotic DNA-replication fidelity and in ensuring correct meiotic recombination. These functions depend on MutL-homolog heterodimers with Mlh1. In humans, MLH1 mutations underlie half of hereditary nonpolyposis colorectal cancers (HNPCCs). Here we report crystal structures of the MutLα (Mlh1-Pms1 heterodimer) C-terminal domain (CTD) from Saccharomyces cerevisiae, alone and in complex with fragments derived from Mlh1 partners. These structures reveal structural rearrangements and additional domains in MutLα as compared to the bacterial MutL counterparts and show that the strictly conserved C terminus of Mlh1 forms part of the Pms1 endonuclease site. The structures of the ternary complexes between MutLα(CTD) and Exo1 or Ntg2 fragments reveal the binding mode of the MIP-box motif shared by several Mlh1 partners. Finally, the structures provide a rationale for the deleterious impact of MLH1 mutations in HNPCCs.

Dual functions of the Hsm3 protein in chaperoning and scaffolding regulatory particle subunits during the proteasome assembly.

The 26S proteasome, a molecular machine responsible for regulated protein degradation, consists of a proteolytic core particle (20S CP) associated with 19S regulatory particles (19S RPs) subdivided into base and lid subcomplexes. The assembly of 19S RP base subcomplex is mediated by multiple dedicated chaperones. Among these, Hsm3 is important for normal growth and directly targets the carboxyl-terminal (C-terminal) domain of Rpt1 of the Rpt1-Rpt2-Rpn1 assembly intermediate. Here, we report crystal structures of the yeast Hsm3 chaperone free and bound to the C-terminal domain of Rpt1. Unexpectedly, the structure of the complex suggests that within the Hsm3-Rpt1-Rpt2 module, Hsm3 also contacts Rpt2. We show that in both yeast and mammals, Hsm3 actually directly binds the AAA domain of Rpt2. The Hsm3 C-terminal region involved in this interaction is required in vivo for base assembly, although it is dispensable for binding Rpt1. Although Rpt1 and Rpt2 exhibit weak affinity for each other, Hsm3 unexpectedly acts as an essential matchmaker for the Rpt1-Rpt2-Rpn1 assembly by bridging both Rpt1 and Rpt2. In addition, we provide structural and biochemical evidence on how Hsm3/S5b may regulate the 19S RP association to the 20S CP proteasome. Our data point out the diverse functions of assembly chaperones.

Structural, thermodynamic, and cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group C protein.

Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 A) X-ray structure of the complex with the peptide N847-R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.

Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones.

Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.

Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking.

The high affinity interaction between the serine protease urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) represents one of the key regulatory steps in cell surface-associated plasminogen activation. On the basis on our crystal structure solved for uPAR in complex with a peptide antagonist, we recently proposed a model for the corresponding complex with the growth factor-like domain of uPA (Llinas et al. (2005) EMBO J. 24, 1655-1663). In the present study, we provide experimental evidence that consolidates and further develops this model using data from a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were determined by surface plasmon resonance. This complete alanine scanning of uPAR highlighted the involvement of 20 surface-exposed side chains in this interaction. Mutations causing delta deltaG > or = 1 kcal/mol for the uPA interaction are all located within or at the rim of the central cavity uniquely formed by the assembly of all three domains in uPAR, whereas none are found outside this crevice. Identification of specific cross-linking sites in uPAR and pro-uPA enabled us to build a model of the uPAR x uPA complex in which the kringle domain of uPA was positioned by the constraints established by the range of these cross-linkers. The nature of this interaction is predominantly hydrophobic and highly asymmetric, thus emphasizing the importance of the shape and size of the central cavity when designing low molecular mass antagonists of the uPAR/uPA interaction.

Structural studies of human placental alkaline phosphatase in complex with functional ligands.

The activity of human placental alkaline phosphatase (PLAP) is downregulated by a number of effectors such as l-phenylalanine, an uncompetitive inhibitor, 5'-AMP, an antagonist of the effects of PLAP on fibroblast proliferation and by p-nitrophenyl-phosphonate (PNPPate), a non-hydrolysable substrate analogue. For the first two, such regulation may be linked to its biological function that requires a reduced and better-regulated hydrolytic rate. To understand how such disparate ligands are able to inhibit the enzyme, we solved the structure of the complexes at 1.6A, 1.9A and 1.9A resolution, respectively. These crystal structures are the first of an alkaline phosphatase in complex with organic inhibitors. Of the three inhibitors, only l-Phe and PNPPate bind at the active site hydrophobic pocket, providing structural data on the uncompetitive inhibition process. In contrast, all three ligands interact at a remote peripheral site located 28A from the active site. In order to extend these observations to the other members of the human alkaline phosphatase family, we have modelled the structures of the other human isozymes and compared them to PLAP. This comparison highlights the crucial role played by position 429 at the active site in the modulation of the catalytic process, and suggests that the peripheral binding site may be involved in the functional specialization of the PLAP isozyme.

Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide.

We report the crystal structure of a soluble form of human urokinase-type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor-stromal microenvironment in many human cancers. The structure was solved at 2.7 A in association with a competitive peptide inhibitor of the urokinase-type plasminogen activator (uPA)-uPAR interaction. uPAR is composed of three consecutive three-finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA-uPAR complex. This model reveals that the receptor-binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA-focalized proteolysis on the cell surface and control cell adhesion and migration.

NAD binding induces conformational changes in Rho ADP-ribosylating clostridium botulinum C3 exoenzyme.

We have solved the crystal structures of Clostridium botulinum C3 exoenzyme free and complexed to NAD in the same crystal form, at 2.7 and 1.95 A, respectively. The asymmetric unit contains four molecules, which, in the free form, share the same conformation. Upon NAD binding, C3 underwent various conformational changes, whose amplitudes were differentially limited in the four molecules of the crystal unit. A major rearrangement concerns the loop that contains the functionally important ARTT motif (ADP-ribosyltransferase toxin turn-turn). The ARTT loop undergoes an ample swinging motion to adopt a conformation that covers the nicotinamide moiety of NAD. In particular, Gln-212, which belongs to the ARTT motif, flips over from a solvent-exposed environment to a buried conformation in the NAD binding pocket. Mutational experiments showed that Gln-212 is neither involved in NAD binding nor in the NAD-glycohydrolase activity of C3, whereas it plays a critical role in the ADP-ribosyl transfer to the substrate Rho. We observed additional NAD-induced movements, including a crab-claw motion of a subdomain that closes the NAD binding pocket. The data emphasized a remarkable NAD-induced plasticity of the C3 binding pocket and suggest that the NAD-induced ARTT loop conformation may be favored by the C3-NAD complex to bind to the substrate Rho. Our structural observations, together with a number of mutational experiments suggest that the mechanisms of Rho ADP-ribosylation by C3-NAD may be more complex than initially anticipated.