Structural Dynamics I: Protein Collective Motions Studied by X-ray Scattering and Diffraction

Conference: 2020: 70th ACA Annual Meeting
08/05/2020: 12:00 PM  - 3:00 PM 
4.1.2 
Oral Session 
Virtual  

Description

The macromolecules of life are often likened to elaborate machines, with many moving parts that must work collectively to achieve biological function. However, it has proven exceedingly difficult to understand how these machines work from traditional, static "snapshots" of structure alone. Thus, a new field of dynamic structural biology has emerged at the intersection of a diverse and evolving set of techniques. In Part I of this two-part session sponsored by Structural Dynamics, we focus on collective motions illuminated by X-ray scattering and diffraction. How are signals transduced within a protein? How are enzymatic activities coordinated in multi-step reactions? Are collective vibrational modes important for activity? This session highlights how cutting-edge X-ray methods, especially time-resolved SAXS/WAXS and crystallography, are providing insights into the dynamic nature of proteins.

Presentations

Opening Remarks

12:00 PM - 12:05 PM 

Time-resolving biomolecular motions on length scales from single Å to Rg

12:05 PM - 12:25 PM 
In this talk I will describe new technologies and new analysis methods that can dramatically enhance our understanding of how biological macromolecules fold and function. Solution scattering, in time-resolved modes, can detect conformational changes on length scales of ~10's of Å. When coupled with methods that embrace the ensemble nature of the time-dependent populations, time-resolved SAXS provides powerful new insight into the rearrangement of backbones of nucleic acids. In conjunction with contrast variation methods, SAXS provides a different perspective on the assembly of protein-nucleic acid complexes. Finally, time-resolved crystallography can now be performed with millisecond resolution, allowing us to follow the Å scale structures of enzymes as they process their substrates. 

View Proposal 327

Author

Lois Pollack, Cornell University Ithaca, NY 

A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly

12:25 PM - 12:45 PM 
Surface layers (S-layers) are crystalline protein coats that encapsulate a variety of bacteria and nearly all archaea. Recent insights into the surface layer protein (SLP) from Caulobacter crescentus, RsaA, have revealed both the high-resolution structure of the S-layer lattice (von Kugelgen et al., Cell 2019) as well as its highly efficient self-assembly via calcium-triggered 2D crystallization (Comerci et al., NComms 2019 and Herrmann et al., Biophys J 2017). However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Utilizing a combination of x-ray crystallography, static and time-resolved small angle x-ray scattering, and time-resolved electron cryo-microscopy (Cryo-EM), we demonstrate that RsaA achieves rapid self-assembly in vitro via multistep crystallization due to sequential changes within the structure and arrangement of protein domains. This assembly pathway involves two domains serving distinct functions. The C-terminal crystallization domain forms the physiological 2D crystal lattice, but the full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain, which also serves to anchor the S-layer to extracellular lipopolysaccharide. Directly observing the RsaA crystallization pathway using a time-course of Cryo-EM imaging revealed a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the two domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Additionally, enhancing the rate of protein crystallization by a discrete nucleation domain unveils possible evolutionary mechanisms to enhance the kinetics of 2D protein crystallization and may enable engineering of kinetically controllable self-assembled macromolecular nanomaterials. 

View Proposal 316

Author

Jonathan Herrmann, Thermo Fisher Scientific Portland, OR 

Additional Author

Soichi Wakatsuki, STANFORD UNIVERSITY Stanford, CA 

Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase

12:45 PM - 1:00 PM 
Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak–trigger–freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme 

View Proposal 272

Author

Yeonoh Shin, The Pennsylvania State University State College, PA 

Additional Author

Katsuhiko Murakami, Penn State Univ University Park, PA 

Coffee Break

1:00 PM - 1:20 PM 

Capturing Reaction Intermediates of the Water Oxidation Reaction In Photosystem II

1:20 PM - 1:45 PM 
The water oxidation reaction in nature occurs in Photosystem II (PS II), multi-subunit protein complex, in which the Mn4CaO5 cluster catalyzes the reaction. The reaction comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at the OEC. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PS II. Using serial femtosecond X-ray crystallography (SFX) and simultaneous X-ray emission spectroscopy (XES) with multi-flash visible laser excitation at room temperature, we have investigated all (meta)stable states. We also collected some timepoint data between the S-states in order to understand the sequence of events. The current status of this research and the mechanistic understanding of the water oxidation reaction based on the X-ray techniques is presented. 

View Proposal 325

Author

Junko Yano, Lawrence Berkeley National Laboratory Berkeley, CA 

Tracking solvent dynamics during carbonic anhydrase catalysis

1:45 PM - 2:00 PM 
Human carbonic anhydrase II (CA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO2/HCO3-. Although CA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. In this presentation, we present the crystallographic structures of CA II cryocooled under various CO2 pressures. The structures reveal new intermediate solvent states of CA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. In addition, the structures provide hints for the role of metal ions in CA II catalysis. 

View Proposal 283

Author

Chae Un Kim, Ulsan National Institute of Science and Technology Ulsan

Time-resolved serial synchrotron crystallography for the functional characterization of proteins

2:00 PM - 2:20 PM 
Functional characterization of proteins requires insights into the interplay between structure and dynamics as a function of time. Classical approaches to X-ray crystallography via populating intermediate states by mutation or chemical trapping only provide static time-averaged data sets. Considering this, serial time-resolved approaches are powerful tools to elucidate time-dependent structures. To this end we combined an established fixed target mount (chip), with a novel "Hit And REturn" (HARE) approach for data acquisition - enabling efficient data collection at microfocus beamlines. With the HARE algorithm a plethora of time delays are accessible from a few milliseconds to several minutes without the burden of an increase in data collection time. Using this approach, we mapped the catalytic cycle of a homodimeric enzyme, fluoroacetate dehalogenase, using a photo-caged substrate and initiating the reaction via laser excitation. A total of 18 time points was collected from 30 milliseconds to 30 seconds capturing key states involved in the reaction pathway. These include substrate binding and reorientation, covalent-intermediate formation, location of the water nucleophile and product release. As well as interactions between the two active sites displayed in molecular breathing and a chain of water molecules modulated by an allosteric pathway. Expanding these approaches with the use of piezo droplet injectors simplifies reaction initiation for systems that are not naturally amenable to light activation. The "Liquid Application Method for time-resolved Analysis" (LAMA) method is very efficient using only minimal amounts of protein (1-3 mg) and ligand (1.5-3 µl) per time point. In combination with the HARE approach and Glucose Isomerase as a model system, we were able to observe substrate binding and an open ring conformation of glucose during its catalytic cycle. These results demonstrate the relevance and applicability of time-resolved approaches to provide insights into the functional dynamics of proteins. 

View Proposal 217

Author

Pedram Mehrabi, Max Planck Institute for the Structure and Dynamics of Matter Hamburg

What's shaking? Molecular-dynamics simulations of X-ray diffraction from crystalline proteins.

2:20 PM - 2:40 PM 
X-ray diffraction data contain rich information about collective motions in protein crystals. Extracting this information has been historically difficult, but advances in data collection and computing are enabling studies of X-ray diffraction from protein crystals using molecular-dynamics (MD) simulations. This talk will examine recent results with a special focus on crystallographic water structure and diffuse scattering -- intensity beneath and away from the Bragg peaks in diffraction images. These results indicate that MD simulations can add realistic descriptions of motions at atomic detail that agree substantially with protein crystallography data. At the same time, limitations in the accuracy of the simulations suggest a path forward for improving MD models and force fields. 

View Proposal 295

Author

MICHAEL WALL, LOS ALAMOS NATIONAL LABORATORY LOS ALAMOS, NM 

Time-resolved serial femtosecond crystallography of the early intermediates in the isopenicillin N synthase reaction with ACV and O2

2:40 PM - 3:00 PM 
The femtosecond pulses at an X-ray free electron laser (XFEL) allow experimental access to enzyme reaction cycles and reveal time-resolved atomic and electronic structures, without X-ray radiation-induced changes to sensitive sites such as an active site metal centre. To this end, our collaboration has developed a drop-on-demand sample delivery system that enables simultaneous collection and correlation of time-resolved femtosecond crystallography (tr-SFX) data with X-ray emission spectroscopy (tr-XES) data.[1] High resolution tr-SFX data yields atomic models throughout the crystals, whereas tr-XES monitors the changes to the electronic structure of the active site metal ion. Isopenicillin N synthase (IPNS) catalyses the nonheme iron-dependant, four electron oxidation of the linear tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into isopenicillin N.[2] A unique feature of the proposed reaction mechanism is the role of two reactive iron species -- an Fe(III)-superoxo and a high-spin Fe(IV)=O species -- that promote the first and second ring closures of the β-lactam, respectively. High valent iron intermediates are of exceptional importance throughout biology where they function as key intermediates, including those that form antimicrobial compounds and in human enzymes, including in hypoxia sensing/response and DNA damage repair.[3] We present results for the early reaction intermediates obtained during O[sub]2[/sub]-catalysed turnover of the IPNS•Fe(II)•ACV complex. Our collaboration has collected dozens of diffraction datasets at several XFELs (LCLS, SACLA, PAL-XFEL) and at Diamond Light Source with room temperature serial crystallography, as well as with freeze-quench cryogenic methods. The room temperature results shown that O[sub]2[/sub] binding to the IPNS•ACV complex results in an ACV-Fe(III)-O-O[sup]-[/sup] complex. As the reaction cycle progresses, conformational changes originating at the iron-ACV complex increase the dynamics of ACV as a result of catalysis. The conformational changes propagate outward to the exterior of the enzyme, where they eventually promote rearrangement of an exterior α-helix with increased thermal parameters. Analogous perturbations to the α-helix are also observed in the anaerobic IPNS•ACV•NO complex under cryogenic conditions, wherein the NO serves as a surrogate for O[sub]2[/sub]. Using solution state [sup]19[/sup]F NMR and specifically labelled protein, we observe increased dynamics of the exterior α-helix in the IPNS•ACV•NO complex. Together, these results indicate several roles for O[sub]2[/sub] biding that influence enzyme and substrate dynamics that ultimately impact the reaction coordinate catalysed at the iron centre by the enzyme. [1] F. D. Fuller, S. Gul, et al, Nat. Methods 14 (2017) 443-449. [2] P. Rabe, J. J. A. G. Kamps, C. J. Schofield, C. T. Lohans, 35 Nat Prod Rep. (2018) 735-756 [3] "2-Oxoglutarate-Dependent Oxygenases", C. J. Schofield & R. P. Hausinger, RSC, Cambridge, 2015, pp. 1–487. Partially supported by a Wellcome Trust Investigator Award in Science (210734/Z/18/Z) and a Royal Society Wolfson Fellowship (RSWF\R2\182017) to AMO. 

View Proposal 291

Author

Allen Orville

Additional Author(s)

Patrick Rabe, University of Oxford Oxford
Jos J. A. G. Kamps, Diamond Light Source Didcot
Cindy Pham, Lawrence Berkeley National Laboratory Berkeley, CA 
Michael McDonough, University of Oxford Oxford
Thomas Leissing, University of Oxford Oxford
Jurgen Brem, University of Oxford Oxford
Pierre Aller, Diamond Light Source Didcot
Agata Butryn, Diamond Light Source Didcot
Franklin Fuller, SLAC National Accelerator Laboratory Menlo Park, CA 
Alex Batyuk, Linac Coherent Light Source, SLAC
Nicholas Sauter, Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Lab
Vittal Sutherlin, Lawrence Berkeley National Laboratory Berkeley, CA 
Junko Yano, Lawrence Berkeley National Laboratory Berkeley, CA 
Jan Kern, Lawrence Berkeley National Laboratory Berkeley, CA 
Christopher Schofield, University of Oxford Oxford