KENNETH C HOLMES. WAYS & MEANS. Solving the structures of macromolecular complexes. Structure 15 July 1994, 2:589-593. The reductionist program for ...
KENNETH C HOLMES KENNETH C HOLMES
WAYS & MEANS WAYS & MEANS
Solving the structures of macromolecular complexes Structure 15 July 1994, 2:589-593
The reductionist program for biology foresees a progression of understanding that will span the gamut from molecules to organisms. Indeed, within a few years the sequencing of genomes will provide us with the canonical inventory of life. To make use of these data we will need to understand protein folding. Equally important, however, is an understanding of the manifold macromolecular interactions which control gene activation and cellular metabolism in their exquisite precision. What is 'understanding': at what level of precision do we need an answer? In the context of explaining cell biology, a 'road map' (Cs-plot) may suffice. Here structural analysis is used as a micro-anatomy to show what is next to what. In favorable cases, such information will provide functional insight, but in many cases a detailed atomic model of the interface between two molecules at about 0.5 A resolution will be necessary to understand their mutual specificity. In general, the necessary prelude to a predictive application of our knowledge will be a truly detailed molecular description of the interaction. This is particularly likely to be the case in medical applications, where one would like to enable rational drug design or complementation by gene therapy. At present, the necessary specificity can only be provided by X-ray structure analysis. Unfortunately, many macromolecular complexes are too large to be tackled by traditional protein crystallographic methods even if they could be persuaded to crystallize. Thus, it would seem that the reductionist program is already judged an intellectual vanity and that biology will long remain descriptive rather than predictive. One possible way out of this dilemma is to 'divide and conquer', a method pioneered by Klug (see [1]). The strategy is to isolate, crystallize, and determine the structures of the components of a macromolecular complex and then combine the structures to form a model of the complex. In general, the way that the structures are combined will be guided by electron microscopy, particularly cryo-electron microscopy (cryoEM), which is widely applicable to macromolecular complexes. Indeed, in cases where the macromolecular complex occurs naturally as a two-dimensional array, cryo-electron microscopy can by itself already yield atomic resolution [2,3]. Unfortunately, many interesting objects won't crystallize but rather form complexes with point-group symmetry or helical symmetry or even no symmetry! These objects do not (or not yet) yield atomic resolution by electron microscopy (EM). Nevertheless, image processing and three-dimensional re-
construction can generally give well-defined electron density maps at 20-30A resolution. The low resolution EM data can then be used to orientate atomic models of single molecules derived from protein crystallography. The combination of electron microscopy with Xray crystallography has been successfully used by a few groups, particularly those working with spherical viruses and muscle. Cryo-EM is considerably more demanding than negative staining but has the advantage that it yields a three-dimensional electron density map, whereas negative staining is limited to visualizing the external surface. As an alternative, fibrous macromolecular complexes sometimes form oriented gels that can yield X-ray fibre diagrams. In some cases, such data may substitute for EM to allow the structure of a macromolecular array to be built up from its component molecules. The following examples illustrate the scope and shortcomings of these different methods.
Actin The structure of monomeric actin (G-actin) was solved by protein crystallography as a 1:1 complex with the enzyme DNase I [4]. Oriented gels of actin fibres (F-actin), a helical copolymer of actin which has 13 molecules in 6 turns repeating every 360A, yield Xray fibre diagrams to about 6A resolution. Using a multi-dimensional search, the orientation of the G-actin monomer which best accounted for the F-actin fibre diagram was determined by comparing observed and calculated fibre diagrams. Fortunately, there is a unique best fit [5] (Fig. 1). The differences remaining reflect the fact that G-actin is not quite F-actin: some (small) conformational changes accompany polymerization. A refinement of the G-actin-based model can be made to give a model for F-actin which provides a perfect fit to the fibre diagram [6] (Fig. 2). Unfortunately, the atomic details are not uniquely defined since the fibre diffraction lacks the appropriate resolution. In particular, the details of the monomer-monomer interactions need to be generated by molecular dynamics annealing of the interfaces using X-PLOR [7]. Comparisons have been made between the atomic model of actin and cryo-EM reconstructions from actin filaments [8,9] (Fig. 3), which agree very well with the model. Moreover, image reconstruction from negative staining can be used to follow small changes in the F-actin structure resulting from changes in the
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polymerization conditions [10]. Negative staining has also been used to show the location of an Fab fragment bound to the charged amino terminus of actin [11].
The actomyosin interaction The interaction of F-actin and the globular head of myosin (myosin subfragment-1 or myosin S1) is cen-
Fig. 1. The structure of F-actin [5]. (a) X-ray fiber diffraction pattern of F-actin. Experimental data are shown in the upper-left and lowerright quadrants. The calculated data from the best fit orientation of the actin monomer are shown in the upper-right and lower-left quadrants. The resolution limit is 8 A. (b) A representation of the atomic model of F-actin. Each molecule of G-actin is shown in a different colour. Each amino acid residue is shown as a small sphere. The green spheres represent residues known to interact with myosin. The molecules are arranged on a helix with 13 molecules in 6 left-handed turns with a repeat every 357.5A. The structure is essentially two long-pitch helices winding slowly round each other. The strong intermolecular contacts are along the long-pitch helices.
Fig. 2. Refined fibre diagram of F-actin [6]. X-ray fibre difraction pattern as in Fig. 1. The calculated quadrants (upperright, lower-left) are now virtually indistinguishable from the experimental data.
Solving the structures of macromolecular complexes Holmes
duce 'decorated actin' in which every actin monomer carries an elongated S1 molecule extending to high radius. Using the 30A resolution cryo-EM reconstruction of decorated actin as a guide [8] and using a standard crystallographic display program (FRODO) [13] it was possible by eye to dock first the F-actin helix and then the elongated S1 molecule into the EM electron density map [14] to yield an atomic model of the actomyosin interaction (Fig. 4). The result was unambiguous. In parallel, a fit was made to a reconstruction obtained from Dictyostelium myosin S1, with essentially the same result [15]. The precision of the result (- 5 A) is to some extent limited by the low resolution of the electron micrograph, but is also affected by the apparent need to change the conformation of the myosin to produce a good fit. Since the model suggests the broad mechanism of muscular contraction [14] it is an example of the usefulness of atomic models of macromolecular complexes even in situations where full atomic precision has not yet been achieved.
Fig. 3. Comparison of the atomic model of F-actin with cryo-EM reconstructions of F-actin [8,91. The two images on the left are derived from the electron microscopy data and are shown at a low-cut electron density level (extreme left) and a high-cut level. The corresponding calculated densities from the atomic model are shown on the right.
tral to an understanding of muscular contraction. The structure of S1 was solved by X-ray crystallography [12]. In the absence of ATP, myosin S1 binds tightly and specifically to F-actin (the rigor complex) to proFig. 4. A stereo pair showing the fitting of atomic models of actin and myosin into the electron density map (shown in green) of 'decorated actin' derived from cryo-EM [14]. The fit of the molecules to the electron density was achieved by 'docking' using the display program FRODO [131. Four subunits of actin are shown (Ca-plot in blue) and one elongated molecule of myosin subfragment 1 (S1 in brown). The myosin interaction site on actin does indeed correspond with the known binding site (see green spheres in Fig. 1). The distal end of the myosin S1 is not in electron density; in fact, the method used to prepare the S1 will have eliminated this part of the molecule.
Flock house virus (FHV) FHV is an icosahedral insect virus having 180 protein subunits arranged in a pseudo-spherical capsid which encloses the genomic RNA. The structure of FHV has been determined by X-ray crystallography [16] allowing the visualization of nearly all the protein capsid but very little of the intemal RNA. The RNA is not visible in the structure because it cannot follow the icosahedral symmetry of the capsid at atomic resolution. In fact, the X-ray structure does show those fragments of duplex RNA that sit under the two-fold axes of the icosahedron and are able to conform to this local symmetry. Threedimensional reconstructions of virus particle images obtained by cryo-EM can, however, show the density of the entire RNA in the interior of the virus (Fig. 5) and can be accurately correlated with the atomic structure of the capsid [17] (Fig. 6). These results demonstrate the quantitative nature of the electron density maps obtained from cryo-EM and their utility as a supplement
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Fig. 5. A reconstruction derived from cryo-electron micrographs of flock house virus (FHV) particles [17]. The T = 3 icosahedral protein capsid is shown in blue. The front surface has been cut away to show the the RNA core (red). Helices from the capsid which interact with the RNA are shown (green).
to X-ray crystallography in determining the structures of macromolecular complexes. Furthermore, the combination of the atomic structure with cryo-EM has been used to characterize an Fab-binding site on the virus [18].
Limitations and prospects The examples cited show that three-dimensional reconstructions of non-crystalline objects embedded in vitreous ice can yield reliable electron density maps. The resolution of such maps (20-30A) is presently limited by the relatively high degrees of underfocus (1-2 mm) required to achieve adequate contrast in the image. This resolution is, however, generally adequate for positioning subunits of known structure. Nevertheless, a serious problem arises in defining details of the interface since all the side chains find themselves in a new environment. Moreover, macromolecules have a strong tendency to indulge in 'induced fit' on mating. This trend has become abundantly clear for protein-DNA complexes [19]. Furthermore, in the case of actin there are two forms, F and G, which differ from each other at atomic resolution, the structure switch being controlled by the polymerization. Moreover, there are 'loops' in actin which only acquire structure in the complex. The structure of these must be computed. This is also true for the actin-myosin interface. The question is how accurately we may be able to predict the structure of a protein-protein interface by calculation if we can define the general way that the proteins fit together using the methods discussed above. A well defined exam-
Fig. 6. (a) The fit of the cryo-electron micrograph reconstruction of FHV [17] (blue) to the atomic model of the capsid [16] around the two-fold axes of the icosahedron. (b) A section through the two-fold axis (centre) and the five-fold axes (sides). Note the perfect agreement between the reconstructed electron density (blue) and the capsid structure (Cac-plot in yellow). The duplex RNA is visible on the two-fold axis (shown as space-filling atomic model). The RNA otherwise does not subscribe to the icosahedral symmetry and is not defined at atomic resolution.
pie, an antigen-antibody complex, has recently been tackled ab initio [20], with some success. If ab initio methods can fold protein loops in more or less well defined environments, we may get what we need. Another possibility is to improve cryo-EM. Here there is indeed hope. In particular the introduction of energyfilter cryo-EM leads to dramatic improvement in the signal to noise ratio in the image [21,22]. This improvement in turn allows images to be obtained closer to focus and therefore with higher resolution. If one can attain 8-10 A resolution from macro-molecular complexes in vitreous ice, a difficult but not impossible task, then one will have enough data to define some elements of secondary structure. This should allow an experimental verification of any 'induced fit' and provide the basis for more constrained, and therefore more precise, attempts to calculate the protein-protein interface.
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Kenneth C Holmes, Max Planck Institut fr medizinische Forschung, 69120 Heidelberg, Germany.
