Insights into biomolecular function from small-angle scattering

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Insights into biomolecular function from small-angle scattering Jill Trewhella Recent advances in neutron and X-ray sources and instrumentation, new and improved scattering techniques, and molecular biology techniques, which have permitted facile preparation of samples, have each led to new opportunities in using small-angle scattering to study the conformations and interactions of biological macromolecules in solution as a function of their properties. For example, new instrumentation on synchrotron sources has facilitated time-resolved studies that yield insights into protein folding. More powerful neutron sources, combined with molecular biology tools that isotopically label samples, have facilitated studies of biomolecular interactions, including those involving active enzymes.

Addresses

Chemical Science and Technology Division, Mail Stop G758, Los Alamos National Laboratory, Los Alamos, NM 87545, USA; e-mail: [email protected] Current Opinion in Structural Biology 1997, 7:?02-?08

http://biomednet.com/elecref/0959440XO0700702 © Current Biology Ltd ISSN 0959-440X Abbreviations

ACTase CaM CD EGF Gla MLCK R state SAXS sEGFR T state TnC Tnl

aspartate transcarbamoylase calmodulin circular dichroism epidermalgrowth factor y-carboxyglutamic acid myosinlight chain kinase relaxedstate small-angleX-ray scattering extraceliular domain of the EGF receptor tense state troponin C troponin I

Introduction

Small-angle scattering of X-rays or neutrons from biological macromolecules in solution yields information on their overall shapes, and the techniques are applicable to molecules and molecular assemblies over a wide range of sizes (10-103A in dimension) [1]. Small-angle scattering data yield parameters such as the radius of gyration, R~, molecular weight, volume, and the vector length distribution function, P(r), of the scattering species. P(r) is the probable frequency of interatomic vector lengths within a scattering particle and is very sensitive to the overall asymmetry and domain structure within the particle. In the case of neutron scattering, deuterium labeling with contrast variation allows one to extract information on the shapes and dispositions of individual components of complex assemblies of biomolecules [2"]. Small-angle scattering is often most powerful when used as a complementary tool with other structural techniques,

such as NMR, crystallography, or electron microscopx; providing the key pieces of information that complete a stor~: Because of its dependence on geometric shape, scattering data can be extremely sensitive to domain orientations and hence to conformational changes and/or flexibilitx, as well as to molecular associations in solution. To extract reliable structural information, solutions of monodisperse, identical particles are ideal, and nonspecific aggregation can be a fatal blow to the small-angle scatterer. T h e ability to work with solutions at low concentrations of solute can alleviate problems of aggregation and is also an advantage for samples difficuh, or expensive to prepare in large quantities. Recently, a number of advances in sources and instrumentation [3,4] have yielded gains in the flux of X-rays or neutrons on samples, which have facilitated more rapid experiments on smaller samples at lower concentrations. Small-angle scattering instrumentation at synchrotron sources (e.g. beam line 4-2 at the Stanford Synchrotron Radiation Laborato~, beam line 15A at the Photon Factory in Tsukuba, instrument D24 at the L U R E - D C I , Orsa,~; France) have made time-resolved studies of protein conformation possible. Recent upgrades of neutron facilities and/or instrumentation (the Cold Neutron Research Facility at the National Institute of Standards Technology recently delivered up to sevenfold increases in neutron fluxes for scattering experiments, and new, more powerful small-angle instrumentation is now available at the Institut Laue-Langevin, Grenoble, France) have resulted in improved small-angle neutron scattering capabilities allowing for studies of relatively small protein complexes (-50 kDa) at less than mg ml -I concentrations. In the area of neutron scattering technique development, Stuhrmann and coworkers [5,6"] have developed a new and potentially powerful method of nuclear-spin contrast variation that increases the signal-to-noise ratio in contrast variation studies, allowing for the location of relatively small components in large complexes, as demonstrated by their determination of the in situ structures of ribosomal proteins [5], and the localization of two tRNAs within the elongating ribosome [6"]. Serdyuk and Zaccai [7] have demonstrated the use of the triple isotopic substitution method with small-angle neutron scattering to extract the scattering profile of one part of a macromolecular complex, determining that this method can characterize a component as small as 2-3% of the total molecular mass. T h e combination of improved scattering instrumentation, new scattering techniques, as well as advances in molecular biology which have permitted facile preparation of pure biological molecules in milligram quantities (including the deuterated form), has significantly increased the power of small-angle scattering to make an impact on structural

Insights into biomolecular function from small-angle scattering Trewhelta

biology research. I will review recent examples of these advances.

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the intact factor X protein so that its biologically active conformation is obtained. Figure 1

P r o b i n g c h a n g e s in s o l u t i o n c o n f o r m a t i o n a s a function of biological properties Domain interactions within a protein Small-angle scattering data are very sensitive to domain orientations and dispositions, which can be key to understanding biomolecular mechanisms. For example, in a small-angle scattering study of the 70kDa heat shock protein DnaK [8], the P(r) functions calculated from the scattering profiles indicate a two-domain structure, consistent with the fact that DnaK has two major functional domains (an N-terminal ATP-binding domain and a C-terminal substrate protein binding domain). Binding of ATP to DnaK results in an overall expansion of the structure, whereas binding of substrate protein results in a decrease in Rg consistent with substrate binding to a cavity or cleft.

More information on domain interactions can be obtained if small-angle solution scattering data can be combined with high-resolution structural data. In the periplasmic receptors for bacterial chemotaxis and transport (maltose-, glucose/galactose- and ribose-binding proteins), crystal structure data on the open, ligand-free [9] and closed, ligand-bound [10] forms of the maltose-binding protein show the two domains rotate using a hinge motion to open and close the ligand-binding cleft that forms between them. Small-angle scattering data [11,12] show that the glucose/galactose- and ribose-binding proteins behave similarly to the maltose-binding protein (as well as the related leucine/isoleucine/valine-binding proteins [13]) in response to ligand binding, which indicates that the hinge action is a general mechanism in this family of proteins. Small-angle scattering also provides an important adjunct to solution N M R structural data, providing critical information on the interactions between domains that may not be well defined by the relatively short-range distance information obtained using NMR. Sunnerhagen et al. [14 °'] have combined N M R and X-ray scattering to study the N-terminal 7-carboxyglutamic acid (Gla) and and epidermal growth factor (EGF) domains of the modular factor X protein which facilitates assembly of constituents of the blood coagulation cascade on membranes at the site of clot formation. T h e Gla domain's affinity for membrane surfaces is Ca2+-dependent. Solution structures for GIa-EGF were obtained from NMR, but the N M R data did not include many interdomain distances, leaving the relative positions of the Gla and EGF domains not well defined. T h e scattering data independently defined the domain orientations and showed unequivocally that the Gla and EGF domains folded toward each other upon Ca 2+ binding to the EGF domain (Figure 1). This reorientation of the domains is postulated to play a key role in orienting

(a)

EGF

(b)

'c~ 199? Current Opinion in Structural Biology

Ccc backbone traces of the Gla-EGF domain pair from the blood coagulation factor X protein. (a) Ca2+-bound form. (b) Ca2+-free form. The structures have been determined by a combination of X-ray scattering and NMR spectroscopy [14"°]. Calcium binding to the EGF consensus site induces a conformational change resulting in the Gla and EGF domains folding toward each other; the Gla domain appears to contribute ligands to the Ca2+-binding site. The Ca 2+ ligands of the Ca 2+ consensus site identified in a high-resolution NMR structure of the isolated EGF domain are shown as ball-and-stick representations.

Quaternary structures Small-angle scattering is of particular value in studying the quaternary structures and conformational changes associated with the biological properties of oligomeric assemblies in solution. Svergun eta/. [15"] have published a small-angle X-ray scattering study showing large differences between crystal and solution quaternary structures of allosteric aspartate transcarbamoylase (ACTase) in its relaxed (R) state. ACTase has six catalytic subunits grouped in two trimers and six regulatory subunits grouped in three dimers, forming a 306 kDa assembly with quasiD3 symmetry. T h e cooperative properties of ACTase activity are accounted for by allosteric interactions based on a reversible transition between a low-affinity tense (T) state and a high-affinity R state. Crystal structures have been obtained for both T and R states showing that the transition involves more or less rigid movements of the subunits such that the distance between the catalytic trimers is increased in the R state [16]. Solution scattering studies show agreement between the solution and crystal forms for the T state, but the R state crystal

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structure does not predict the solution scattering profiles. A model has been developed to better fit the scattering data by allowing significantly larger movements of the catalytic subunits (Figure 2). This study demonstrates that the ability to evaluate the solution structures of large oligomeric structures is of critical importance, particularly when these function via a transition between structural forms. Protein-protein and protein-DNA interactions

Small-angle scattering can be powerful for probing interactions between macromolecules in solution. For example, small-angle X-ray and neutron scattering using specific deuterium labeling have found rich territory in the developing story of CaZ+-binding proteins and their interactions with regulatory targets (for a review, see [17]). T h e dumbbell-shaped protein calmodulin (CAM) activates a diverse array of target enzymes, many of which have CaM-binding domains consisting of short ( - 2 0 residue) sequences that have a high propensity for forming an amphipathic helix with two large hydrophobic residues (spaced by - 12 residues) which interact with the hydrophobic clefts in each of CaM's lobes (for reviews, see [18,19]). To date, structural studies of CaM-target enzyme interactions have focused primarily on these short helical target peptides. Small-angle neutron scattering experiments using deuterium labeling and contrast variation have recently revealed the first structural information on CaM complexed with a catalytically active myosin light chain kinase (MLCK; Figure 3; [20"]). The scattering data show that CaM undergoes an unhindered conformational collapse upon binding MLCK which is very similar to that observed with the isolated CaM-binding peptides. Further, the scattering data support the autoinhibitory

hypothesis for M L C K activation [21] by indicating CaM binding to the enzyme induces a significant movement of the kinase's CaM-binding and autoinhibitory sequences away from the surface of the catalytic core. This rest, It is in stark contrast to the earlier neutron scattering studies of the muscle protein troponin C (TnC) and its interaction with troponin I (TnI) [22]. T n C is evolutionarilv related to CaM and shares many structural and functional similarities. T h e scattering data show, however, that T n C remains extended in its interaction with TnI, exerting its regulatory role through the Cae+-dependent interaction between the N-terminal lobe of T n C and TnI that modulates TnI's interaction with actin molecules within the thin filaments of muscle. Small-angle X-ray scattering, combined with titration calorimetry [23"], has been used to show that EGF induces a quantitative formation of dimers of the extracellular domain of the EGF receptor (sEGFR). The induction of receptor oligomerization by ligand binding is the first step in the activation of growth factor receptors, as well as of other cytokine receptors anchored to their membranes by a single c* helix. T h e data suggest s E G F R dimerization first involves the formation of the two E G F : s E G F R monomers, which associate either via bivalent binding of the EGF ligands or via a ligand-induced conformational change that opens up sites for interaction. The bivalent model for the dimerization suggests possible mechanisms for both homo- and heterodimcrization within this family of receptors. Coleman and coworkers [24] have used small-angle X-ray scattering to identify a novel dimerization domain in the RAG1 protein, characterizing the overall shape of the dimerization domain and determining the orientations of the monomeric subunits in the dimer. RAG1 proteins play a central role in the earliest stages

Figure 2

3nm

Models of aspartate transcarbamoylase quaternary structure. Darkly shaded subunits (top and bottom) represent the two catalytic trimers; lightly shaded subunits (equatorial) represent the three regulatory dimers. The enzyme functions via an allosteric mechanism involving a reversible transition between a low-affinity T and a high-affinity R state. T and Rc are derived from crystal structure data, whereas Rs is a model constructed to fit the small-angle solution scattering data [15"]. The major difference between the T and Rc structures is seen in the relative rotations of the catalytic subunRs. The solution data indicate a significantly larger rotation of the catalytic subunits in the R state. Adapted with permission from [15"°].

Insights into biomolecular function from small-angle scattering Trewhella

Figure 3

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and sometimes diversity (D) gene segments to produce the genetic sequence encoding the variable region of the antigen-binding proteins. The dimerization domain consists of two zinc-binding motifs (a C3HC4 zinc ring finger subdomain and a C2H2 zinc finger sequence). T h e position of the dimerization domain immediately adjacent to the core region of RAG1 is consistent with it serving an important function in V(D)J recombination. Tuzikov et al. [25] have published a small-angle scattering study of a two-step equilibrium enzyme-substrate interaction and have developed methods for extracting the stoichiometry, equilibrium, and structural parameters of the enzyme-substrate complex. T h e Eco dam methyltransferase enzyme recognizes a specific double-stranded DNA sequence (GATC), binding in a 2:1 enzyme:substrate stoichiometric ratio. Using scattering data from samples of the enzyme (E) and substrate (S) in different molar ratios, Tuzikov et al. [25] demonstrated they could calculate the scattering profiles for the ES and E2S complexes and derive the structural parameters for the enzyme, the substrate and their complexes.

Model structure of CaM complexed with MLCK. The model is derived from neutron scattering studies of deuterated CaM complexed with a catalytically active MLCK [20"]. The basic scattering functions for the individual protein components are extracted from neutron scattering data measured for the complex in a series of solvents with differing D20 levels. The basic scattering functions define the individual shapes and dispositions of the components which are represented by crosses. The high-resolution structures of CaM complexed with the 20-residue MLCK-I helical peptide (bottom) [35], and of the conserved catalytic core of the kinase (top; based on the cAMP-dependent protein kinase structure [36]) are fit into the ellipsoid shapes derived from the scattering data. The upper and lower lobes of the catalytic core of MLCK are represented as gray and black ribbon drawings, respectively, with the catalytic cleft between them labeled. The empty spaces in the ellipsoid representing MLCK are occupied by its N- and C-terminal sequence segments, the structures of which are unknown at this time. CaM is represented as a gray ribbon drawing, with its bound MLCK-I peptide in black; a tryptophan residue near the N-terminal end which is believed to be key to CaM recognition is shown as a space-filling model. The autoinhibitory sequence of MLCK forms an extensive network of contacts with the surface of the catalytic core in the absence of CaM [37]. The autoinhibitory sequence (-25 residues) overlaps with the N-terminal residues of the CaM-binding sequence. The position of CaM with respect to the kinase in the model thus indicates that a significant movement of the autoinhibitory sequence away from the surface of the catalytic core must occur upon CaM binding. Adapted with permission from [20°°].

of V(D)J recombination - - a site-specific recombination event that selectively combines variable (V), joining (J)

Protein folding Equilibrium studies T h e compactness of a protein structure is an important parameter that characterizes its degree of folding. Smallangle scattering is one of the few direct means by which to measure compactness and determine information on the geometric size and shape of the molecule. Scattering can be used therefore as a simple check of the structural integrity of a folded protein, for example, as a function of modification by mutagenesis [26]. As protein unfolding often involves the exposure of hydrophobic groups, protein aggregation can be a limiting factor in the utility of scattering techniques for probing compactness in unfolded structures; hence, the utilization of synchrotron X-ray intensities can be a great advantage in facilitating working at the lowest protein concentrations.

By combining small-angle scattering and circular dichroism (CD) data, Doniach and coworkers [27"'] discovered a partially folded lysozyme intermediate that was determined to be relatively compact, but with a C D signature suggesting some random coil component. Their scattering data also clearly supported a nonrandom coil structure in the denatured state of lysozyme, even in 8 M urea at pH 2.9. Doniach and coworkers [27 °°] have been able to separate the scattering profiles of the native and unfolded states from the putative partially folded intermediate using data acquired at different concentrations of urea. From these scattering profiles, they calculated P(r) for each form, clearly showing the relative degree of compaction in each (Figure 4). A model of the intermediate was developed on the basis of a kinetic intermediate consisting of a compact molten globule domain and a disordered region that had been identified in the refolding pathway of lysozyme using IH/ZH exchange data [28].

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and heat denatured forms in terms of different folding pathways for the two forms of the protein. Castellano et al. [30] propose that the ovalbumin to S-ovalbumin transformation is actually a protein switch triggered by changes in the chemical environment.

Figure 4

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I 10

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I 50

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r(A) Vector length distribution functions, P(r) versus r, for lysozyme in different folded states. The P(r) profiles are derived from small-angle scattering data from lysozyme in its native state (short dash), its intermediate folded state extracted from scattering data obtained as a function of denaturant concentration (long dash), and its 'unfolded' state in 8 M urea (solid line) [27°°]. The open circles represent P(r) calculated from the crystal structure of lysozyme, which show good agreement with that determined from the solution scattering data on the native state. The P(r) profiles show the protein progressively becoming less compact as it transitions from its native state through the intermediate state to the unfolded state: the peak of the P(r) progressively shifts to longer vectors lengths, and the number of vector lengths >35)~ also increases. Adapted with permission from [27°'].

In another equilibrium 'unfolding' study, Regan and coworkers [29 °] undertook a combined CD, fluorescence, calorimetry, and scattering study of surface point mutations of the B1 domain of streptococcal protein G aimed at evaluating the structure and stability of the mutant proteins' denatured states. Small-angle scattering data revealed differences in the average degree of compaction seen as a function of denaturant concentration for different mutants. The least stable mutant showed increased Rg values for the apparent denatured state (compared with the native folded state) at guanadinium hydrochloride concentrations as low as 0.5 M, whereas >2 M guanadinium hydrochloride was required to see the same effect in the most stable mutant. These observations were consistent with denaturation curves determined using CD and provided the first example of a direct observation of an alteration in the structure of the denatured ensemble in response to increasing denaturant concentration. Castellano et al. [30] have used small-angle scattering to examine the solution conformations of ovalbumin and S-ovalbumin, two states of the same protein proposed by some to be an example of a protein that exists in a distinct metastable state (ovalbumin) that is somehow transformed to a lower energy state (S-ovalbumin). T h e y interpreted small-angle scattering data on native ovalbumin and S-ovalbumin, as well as on their own chemically denatured

Scattering applications to studying protein unfolding require the development of methods to adequately model the unfolded states. Smith and coworkers [31] have published a method for calculating the scattering profile of a strongly denatured protein that exists as a configurational distribution rather than as a well-defined subset of closely related structures. Their modeling approach utilizes freely jointed chains of spheres and compares well with small-angle neutron scattering data from strongly denatured phosphoglycerate kinase, as well as with a rapid method for calculating scattering profiles from a full all-atom configuration of a denatured protein. Time-resolved studies

Improved X-ray intensities from synchrotron sources have made time-resolved studies of protein folding possible [32]. T h e challenge has been to increase the timeresolution into the millisecond regime. Semisotnov and colleagues [33 °] have monitored protein globularization during folding using a parameter they define as the 'integrated SAXS intensity' which is simply the total scattering intensity integrated over a finite measurement range of scattering angle and hence does not require the high-precision measurement of the scattering profile needed for determination of Rg or P(r). T h e integrated SAXS intensity parameter can thus be determined relatively rapidly, and it is also less sensitive to protein aggregation effects than Rg values. Time-resolved scattering data were measured using stopped-flow methods to obtain denaturant-induced protein unfolding, and these data were compared with equilibrium measurements, as well as with theoretical calculations on globular and unfolded structures. Distinctive behavior in terms of globularization has been observed between bovine carbonic anhydrase B and yeast phosphoglycerate kinase during folding. Conclusions

Further advances in X-ray and neutron sources, instruments, and technique development are continuing to expand the possibilities for small-angle scattering as a structural biology tool. With the Advanced Photon Source at Argonne National Laboratory coming online and promising even greater X-ray intensities than are currently available in the US, we will be able to make measurements even more rapidly and/or with less material. T h e increased beam intensities will expand the potential for time-resolved studies, especially if faster methods than stopped flow are used for triggering reactions (e.g. T-jump methods or caged compounds). Further developments in neutron sources, such as the upgrade of the short-pulsed spallation source at Los Alamos and the planned next generation spallation source at Oak Ridge,

Insights into b i o m o l e c u l a r function f r o m small-angle scattering Trewhella

will provide new capabilities for small-angle scattering in the US, including an increased potential for using neutron resonance scattering as a structural biology tool [34"]. T h e s e developments should serve to increase the accessibility and utility of small-angle scattering techniques for studying biomolecular conformations and interactions in solution as a function of biological properties over a broad range of dimensions.

Acknowledgements b,l.v thanks to Jinkui Zhao and Joanna Kruegcr for critically reading this manuscript, and to Sebastian Doniach and Dimitri Svcrgun for kindly making available figures for this review.

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