Robert P. Rambo

Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

Structural mechanism of abscisic acid binding and signaling by dimeric PYR1.

ABA Receptor Up Close Plants face a variety of environmental stresses, including drought, salinity, and cold. In the face of such stresses, the plant hormone abscisic acid (ABA) triggers adaptive physiological responses. Nishimura et al. (p. 1373, published online 22 October; see the Perspective by Sussman and Phillips) have now analyzed the crystal structure of one member of the ABA receptor family, PYR1 (pyrabactin resistance 1). The ABA molecule binds within an internal pocket of PYR1, where it probably induces a conformational change. The plant hormone responsible for drought tolerance signals by inducing conformational changes in its dimeric protein receptor. The phytohormone abscisic acid (ABA) acts in seed dormancy, plant development, drought tolerance, and adaptive responses to environmental stresses. Structural mechanisms mediating ABA receptor recognition and signaling remain unknown but are essential for understanding and manipulating abiotic stress resistance. Here, we report structures of pyrabactin resistance 1 (PYR1), a prototypical PYR/PYR1-like (PYL)/regulatory component of ABA receptor (RCAR) protein that functions in early ABA signaling. The crystallographic structure reveals an α/β helix–grip fold and homodimeric assembly, verified in vivo by coimmunoprecipitation. ABA binding within a large internal cavity switches structural motifs distinguishing ABA-free “open-lid” from ABA-bound “closed-lid” conformations. Small-angle x-ray scattering suggests that ABA signals by converting PYR1 to a more compact, symmetric closed-lid dimer. Site-directed PYR1 mutants designed to disrupt hormone binding lose ABA-triggered interactions with type 2C protein phosphatase partners in planta.

Accurate assessment of mass, models and resolution by small-angle scattering

Modern small-angle scattering (SAS) experiments with X-rays or neutrons provide a comprehensive, resolution-limited observation of the thermodynamic state. However, methods for evaluating mass and validating SAS-based models and resolution have been inadequate. Here we define the volume of correlation, Vc, a SAS invariant derived from the scattered intensities that is specific to the structural state of the particle, but independent of concentration and the requirements of a compact, folded particle. We show that Vc defines a ratio, QR, that determines the molecular mass of proteins or RNA ranging from 10 to 1,000 kilodaltons. Furthermore, we propose a statistically robust method for assessing model-data agreements (χ2free) akin to cross-validation. Our approach prevents over-fitting of the SAS data and can be used with a newly defined metric, RSAS, for quantitative evaluation of resolution. Together, these metrics (Vc, QR, χ2free and RSAS) provide analytical tools for unbiased and accurate macromolecular structural characterizations in solution.

Characterizing Flexible and Intrinsically Unstructured Biological Macromolecules by SAS Using the Porod-Debye Law

Unstructured proteins, RNA or DNA components provide functionally important flexibility that is key to many macromolecular assemblies throughout cell biology. As objective, quantitative experimental measures of flexibility and disorder in solution are limited, small angle scattering (SAS), and in particular small angle X-ray scattering (SAXS), provides a critical technology to assess macromolecular flexibility as well as shape and assembly. Here, we consider the Porod-Debye law as a powerful tool for detecting biopolymer flexibility in SAS experiments. We show that the Porod-Debye region fundamentally describes the nature of the scattering intensity decay by capturing the information needed for distinguishing between folded and flexible particles. Particularly for comparative SAS experiments, application of the law, as described here, can distinguish between discrete conformational changes and localized flexibility relevant to molecular recognition and interaction networks. This approach aids insightful analyses of fully and partly flexible macromolecules that is more robust and conclusive than traditional Kratky analyses. Furthermore, we demonstrate for prototypic SAXS data that the ability to calculate particle density by the Porod-Debye criteria, as shown here, provides an objective quality assurance parameter that may prove of general use for SAXS modeling and validation.

Structure of the SAM-II riboswitch bound to S -adenosylmethionine

In bacteria, numerous genes harbor regulatory elements in the 5′ untranslated regions of their mRNA, termed riboswitches, which control gene expression by binding small-molecule metabolites. These sequences influence the secondary and tertiary structure of the RNA in a ligand-dependent manner, thereby directing its transcription or translation. The crystal structure of an S-adenosylmethionine–responsive riboswitch found predominantly in proteobacteria, SAM-II, has been solved to reveal a second means by which RNA interacts with this important cellular metabolite. Notably, this is the first structure of a complete riboswitch containing all sequences associated with both the ligand binding aptamer domain and the regulatory expression platform. Chemical probing of this RNA in the absence and presence of ligand shows how the structure changes in response to S-adenosylmethionine to sequester the ribosomal binding site and affect translational gene regulation.

Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.

Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element

Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8Å resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.

SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication

SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1) maintains genome integrity during DNA replication. Here we investigated its mechanism of action. We found that SMARCAL1 travels with elongating replication forks, and its absence leads to MUS81-dependent double-strand break formation. Binding to specific nucleic acid substrates activates SMARCAL1 activity in a reaction that requires its HARP2 (Hep-A-related protein 2) domain. Homology modeling indicates that the HARP domain is similar in structure to the DNA-binding domain of the PUR proteins. Limited proteolysis, small-angle X-ray scattering, and functional assays indicate that the core enzymatic unit consists of the HARP2 and ATPase domains that fold into a stable structure. Surprisingly, SMARCAL1 is capable of binding three-way and four-way Holliday junctions and model replication forks that lack a designed ssDNA region. Furthermore, SMARCAL1 remodels these DNA substrates by promoting branch migration and fork regression. SMARCAL1 mutations that cause Schimke immunoosseous dysplasia or that inactivate the HARP2 domain abrogate these activities. These results suggest that SMARCAL1 continuously surveys replication forks for damage. If damage is present, it remodels the fork to promote repair and restart. Failures in the process lead to activation of an alternative repair mechanism that depends on MUS81-catalyzed cleavage of the damaged fork.

Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering

Small-angle X-ray scattering (SAXS) is changing how we perceive biological structures, because it reveals dynamic macromolecular conformations and assemblies in solution. SAXS information captures thermodynamic ensembles, enhances static structures detailed by high-resolution methods, uncovers commonalities among diverse macromolecules, and helps define biological mechanisms. SAXS-based experiments on RNA riboswitches and ribozymes and on DNA-protein complexes including DNA-PK and p53 discover flexibilities that better define structure-function relationships. Furthermore, SAXS results suggest conformational variation is a general functional feature of macromolecules. Thus, accurate structural analyses will require a comprehensive approach that assesses both flexibility, as seen by SAXS, and detail, as determined by X-ray crystallography and NMR. Here, we review recent SAXS computational tools, technologies, and applications to nucleic acids and related structures.

Super-Resolution in Solution X-Ray Scattering and Its Applications to Structural Systems Biology

Small-angle X-ray scattering (SAXS) is a robust technique for the comprehensive structural characterizations of biological macromolecular complexes in solution. Here, we present a coherent synthesis of SAXS theory and experiment with a focus on analytical tools for accurate, objective, and high-throughput investigations. Perceived SAXS limitations are considered in light of its origins, and we present current methods that extend SAXS data analysis to the super-resolution regime. In particular, we discuss hybrid structural methods, illustrating the role of SAXS in structure refinement with NMR and ensemble refinement with single-molecule FRET. High-throughput genomics and proteomics are far outpacing macromolecular structure determinations, creating information gaps between the plethora of newly identified genes, known structures, and the structure-function relationship in the underlying biological networks. SAXS can bridge these information gaps by providing a reliable, high-throughput structural characterization of macromolecular complexes under physiological conditions.