Nucleic acid aptamers selected through systematic evolution of ligands by exponential enrichment (SELEX) fold into exquisite globular structures in complex with protein targets with diverse translational applications. Varying the chemistry of nucleotides allows evolution of nonnatural nucleic acids, but the extent to which exotic chemistries can be integrated into a SELEX selection to evolve nonnatural macromolecular binding interfaces is unclear. Here, we report the identification of a cubane-modified aptamer (cubamer) against the malaria biomarker Plasmodium vivax lactate dehydrogenase (PvLDH). The crystal structure of the complex reveals an unprecedented binding mechanism involving a multicubane cluster within a hydrophobic pocket. The binding interaction is further stabilized through hydrogen bonding via cubyl hydrogens, previously unobserved in macromolecular binding interfaces. This binding mechanism allows discriminatory recognition of P. vivax over Plasmodium falciparum lactate dehydrogenase, thereby distinguishing these highly conserved malaria biomarkers for diagnostic applications. Together, our data demonstrate that SELEX can be used to evolve exotic nucleic acids bearing chemical functional groups which enable remarkable binding mechanisms which have never been observed in biology. Extending to other exotic chemistries will open a myriad of possibilities for functional nucleic acids.

Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.

Melatonin is a synchronizer of many physiological processes. Abnormal melatonin signaling is associated with human disorders related to sleep, metabolism, and neurodevelopment. Here, we present the X-ray crystal structure of human N-acetyl serotonin methyltransferase (ASMT), the last enzyme of the melatonin biosynthesis pathway. The polypeptide chain of ASMT consists of a C-terminal domain, which is typical of other SAM-dependent O-methyltransferases, and an N-terminal domain, which intertwines several helices with another monomer to form the physiologically active dimer. Using radioenzymology, we analyzed 20 nonsynonymous variants identified through the 1000 genomes project and in patients with neuropsychiatric disorders. We found that the majority of these mutations reduced or abolished ASMT activity including one relatively frequent polymorphism in the Han Chinese population (N17K, rs17149149). Overall, we estimate that the allelic frequency of ASMT deleterious mutations ranges from 0.66% in Europe to 2.97% in Asia. Mapping of the variants on to the 3-dimensional structure clarifies why some are harmful and provides a structural basis for understanding melatonin deficiency in humans.

The Plasmodium subtilase SUB1 plays a pivotal role during the egress of malaria parasites from host hepatocytes and erythrocytes. Here we report the crystal structure of full-length SUB1 from the human-infecting parasite Plasmodium vivax, revealing a bacterial-like catalytic domain in complex with a Plasmodium-specific prodomain. The latter displays a novel architecture with an amino-terminal insertion that functions as a 'belt', embracing the catalytic domain to further stabilize the quaternary structure of the pre-protease, and undergoes calcium-dependent autoprocessing during subsequent activation. Although dispensable for recombinant enzymatic activity, the SUB1 'belt' could not be deleted in Plasmodium berghei, suggesting an essential role of this domain for parasite development in vivo. The SUB1 structure not only provides a valuable platform to develop new anti-malarial candidates against this promising drug target, but also defines the Plasmodium-specific 'belt' domain as a key calcium-dependent regulator of SUB1 during parasite egress from host cells.

Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by HO leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete , a rare example of PerR lacking a structural metal-binding site. studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion., leptospiral PerR could bind to the promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA-binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress.

In many γ-proteobacteria, the RpoS/σS sigma factor associates with the core RNAP (RNA polymerase) to modify global gene transcription in stationary phase and under stress conditions. The small regulatory protein Crl stimulates the association of σS with the core RNAP in Escherichia coli and Salmonella enterica serovar Typhimurium, through direct and specific interaction with σS. The structural determinants of Crl involved in σS binding are unknown. In the present paper we report the X-ray crystal structure of the Proteus mirabilis Crl protein (CrlPM) and a structural model for Salmonella Typhimurium Crl (CrlSTM). Using a combination of in vivo and in vitro assays, we demonstrated that CrlSTM and CrlPM are structurally similar and perform the same biological function. In the Crl structure, a cavity enclosed by flexible arms contains two patches of conserved and exposed residues required for σS binding. Among these, charged residues that are likely to be involved in electrostatic interactions driving Crl-σS complex formation were identified. CrlSTM and CrlPM interact with domain 2 of σS with the same binding properties as with full-length σS. These results suggest that Crl family members share a common mechanism of σS binding in which the flexible arms of Crl might play a dynamic role.

Pathogenic Leptospira spp. are the agents of leptospirosis, an emerging zoonotic disease. Analyses of Leptospira genomes have shown that the pathogenic leptospires (but not the saprophytes) possess a large number of genes encoding proteins containing leucine-rich repeat (LRR) domains. In other pathogenic bacteria, proteins with LRR domains have been shown to be involved in mediating host-cell attachment and invasion, but their functions remain unknown in Leptospira. To gain insight into the potential function of leptospiral LRR proteins, the crystal structures of four LRR proteins that represent a novel subfamily with consecutive stretches of a 23-amino-acid LRR repeat motif have been solved. The four proteins analyzed adopt the characteristic α/β-solenoid horseshoe fold. The exposed residues of the inner concave surfaces of the solenoid, which constitute a putative functional binding site, are not conserved. The various leptospiral LRR proteins could therefore recognize distinct structural motifs of different host proteins and thus serve separate and complementary functions in the physiology of these bacteria.

The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example.