EF-hand Protein Research Articles

Structural, Thermodynamic, and Cellular Characterization of Human Centrin 2 Interaction with Xeroderma Pigmentosum Group C Protein

Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847–R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 Å) X-ray structure of the complex with the peptide N847–R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.

Energetics of the Native Energy Landscape of a Two-Domain Calcium Sensor Protein: Distinct Folding Features of the Two Domains

The protein folding energy landscape allows a thorough understanding of the protein folding problem which in turn helps in understanding various aspects of biological functions. Characterizing the cooperative unfolding units and the intermediates along the folding funnel of a protein is a challenging task. In this paper, we investigated the native energy landscape of EhCaBP, a calcium sensor, belonging to the same EF-hand superfamily as calmodulin. EhCaBP is a two-domain EF-hand protein consisting of two EF-hands in each domain and binding to four Ca2+ cations. Native-state hydrogen exchange (HX) was used to assess the folding features of the landscape and also to throw light on the structure-folding function paradigm of calcium sensor proteins. HX measurements under the EX2 regime provided the thermodynamic information about the protein folding events under native conditions. HX studies revealed that the unfolding of EhCaBP is not a two-state process. Instead, it proceeds through cooperative units. The C-terminal domain exhibits less denaturant dependence than the N-terminal domain, suggesting that the former is dominated by local fluctuations. It is interesting to note that the N- and C-terminal domains of EhCaBP have distinct folding features. In fact, these observed differences can regulate the domain-dependent target recognition of two-domain Ca2+ sensor proteins.

Centrin controls the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels Ca2+-dependently

In Paramecium, ciliary reversal is coupled with voltage-gated Ca2+ channels on the ciliary membrane. We previously isolated a P. caudatum mutant, cnrC, with a malfunction of the Ca2+ channels and discovered that the channel activity of cnrC was restored by transfection of the P. caudatum centrin (Pccentrin1p) gene, which encodes a member of the Ca2+-binding EF-hand protein family. In this study, we injected various mutated Pccentrin1p genes into cnrC and investigated whether these genes restore the Ca2+ channel activity of cnrC. A Pccentrin1p mutant gene lacking Ca2+ sensitivity of the third and fourth EF-hands lost the ability to restore the channel function of cnrC, and mutation of the fourth EF-hand caused more serious impairment than mutation of the third EF-hand. Moreover, a Pccentrin1p gene lacking the N-terminal 34-amino acid sequence also lost the ability to restore the channel activity. Native-PAGE analysis demonstrated that the N-terminal sequence is important for the Ca2+-dependent structural change of Pccentrin1p. These results demonstrate that Pccentrin1p Ca2+-dependently regulates the Ca2+ channel activity in vivo.

Structural and functional insights into RAGE activation by multimeric S100B

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF-hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X-ray structure of human Ca(2+)-loaded S100B was determined at 1.9 A resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size-exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V-domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation.

Structures and metal-ion-binding properties of the Ca2+-binding helix–loop–helix EF-hand motifs

The 'EF-hand' Ca2+-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix-loop-helix secondary structure as well as the ligands presented by the loop to bind the Ca2+ ion. The identity of these ligands is semi-conserved in the most common (the 'canonical') EF-hand; however, several non-canonical EF-hands exist that bind Ca2+ by a different co-ordination mechanism. EF-hands tend to occur in pairs, which form a discrete domain so that most family members have two, four or six EF-hands. This pairing also enables communication, and many EF-hands display positive co-operativity, thereby minimizing the Ca2+ signal required to reach protein saturation. The conformational effects of Ca2+ binding are varied, function-dependent and, in some cases, minimal, but can lead to the creation of a protein target interaction site or structure formation from a molten-globule apo state. EF-hand proteins exhibit various sensitivities to Ca2+, reflecting the intrinsic binding ability of the EF-hand as well as the degree of co-operativity in Ca2+ binding to paired EF-hands. Two additional factors can influence the ability of an EF-hand to bind Ca2+: selectivity over Mg2+ (a cation with very similar chemical properties to Ca2+ and with a cytoplasmic concentration several orders of magnitude higher) and interaction with a protein target. A structural approach is used in this review to examine the diversity of family members, and a biophysical perspective provides insight into the ability of the EF-hand motif to bind Ca2+ with a wide range of affinities.

Calcium‐induced folding of a fragment of calmodulin composed of EF‐hands 2 and 3

Calmodulin (CaM) is an EF-hand protein composed of two calcium (Ca(2+))-binding EF-hand motifs in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). In this study, we examined the structure, dynamics, and Ca(2+)-binding properties of a fragment of CaM containing only EF-2 and EF-3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca(2+)-free CaM2/3 is predominantly unfolded, but upon binding Ca(2+), adopts a monomeric structure composed of two EF-hand motifs bridged by a short antiparallel beta-sheet. Despite having an "even-odd" pairing of EF-hands, the tertiary structure of CaM2/3 is similar to both the "odd-even" paired N- and C-domains of Ca(2+)-ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter-EF-hand loop. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca(2+) binding with a K (d1) = 30 +/- 5 microM to EF-3, and a K (d2) > 1000 microM to EF-2. Binding of the first equivalent of Ca(2+) induces the cooperative folding of CaM2/3. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF-hand domain together, while the loop between EF-hands covalently tethers the second and third helices. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its "even-odd" paired EF-hands. This nonnative hydrophobic core packing may contribute to the weak Ca(2+) affinity exhibited by EF-2 in the context of CaM2/3.

The Impact of X-ray Crystallography and NMR on Intracellular Calcium Signal Transduction by EF-Hand Proteins: Crossing the Threshold from Structure to Biology and Medicine

The use of x-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy has revolutionized our understanding of the transduction of intracellular calcium signals into cellular responses. Indeed, the ability to cross a critical threshold from basic descriptive structural biochemistry to real-world applications in therapeutics and biotechnology now looms on the experimental horizon. The ~500 atomic resolution structures determined by x-ray and NMR approaches and deposited in the Protein Data Bank, many of which are of complexes of EF-hand proteins with peptide fragments of cellular targets, have yielded an extremely thorough description for how EF-hand proteins respond to the binding of calcium. Although this database of structures is a powerful structural tool to describe EF-hand protein function, it is limited in its ability to have a significant impact on biology and medicine because the structural effects on the downstream target are not determined. The opportunity that now lies before us is to extend this EF-hand-centric structural information so that the alterations in the target proteins are defined and the structural basis for functional consequences downstream is understood.

Domain Stability and Metal-Induced Folding of Calcium- and Integrin-Binding Protein 1

It is widely accepted that a pair of EF-hands is the functional unit of typical four EF-hand proteins such as calmodulin or troponin C. In this work we investigate the structure and stability of the four EF-hand domains in the related protein calcium- and integrin-binding protein 1 (CIB1) in the presence and absence of Mg2+ or Ca2+, to determine if similar EF-hand interactions occur. The backbone structure and flexibility of CIB1 were first studied by NMR spectroscopy, and these studies were complimented with steady-state fluorescence spectroscopy and chemical denaturation experiments using mutant CIB1 proteins having single Trp reporter groups in each of the four EF-hand domains EF-I (F34W), EF-II (F91W), EF-III (L128W), and EF-IV (F173W). We find that Mg2+-CIB1 adopts a well-folded structure similar to Ca2+-CIB1, except for some conformational heterogeneity in the C-terminal EF-IV domain. The structure of apo-CIB1 is significantly more dynamic, especially within EF-II, EF-III, and a partially unfolded EF-IV region, but the N-terminal EF-I region of apo-CIB1 has a well-ordered and more stable structure. The data reveal significant communication between the N- and C-lobes of CIB1, and show that transient intermediate conformations are formed along the unfolding pathway for each form of the protein. Collectively the data demonstrate that the communication between the paired EF-hand domains as well as between the N- and C-lobes of CIB1 is distinct from the ancestral proteins calmodulin and troponin C, which might be important for the unique function of CIB1 in numerous biological processes.

The Crystal Structure of the Human (S100A8/S100A9) 2 Heterotetramer, Calprotectin, Illustrates how Conformational Changes of Interacting α-Helices Can Determine Specific Association of Two EF-hand Proteins

The EF-hand proteins S100A8 and S100A9 are important calcium signalling proteins that are involved in wound healing and provide clinically relevant markers of inflammatory processes, such as rheumatoid arthritis and inflammatory bowel disease. Both can form homodimers via distinct modes of association, probably of lesser stability in the case of S100A9, whereas in the presence of calcium S100A8 and S100A9 associate to calprotectin, the physiologically active heterooligomer. Here we describe the crystal structure of the (S100A8/S100A9) 2 heterotetramer at 1.8 Å resolution. Its quaternary structure illustrates how specific heteroassociation is energetically driven by a more extensive burial of solvent accessible surface areas in both proteins, most pronounced for S100A9, thus leading to a dimer of heterodimers. A major contribution to tetramer association is made by the canonical calcium binding loops in the C-terminal halves of the two proteins. The mode of heterodimerisation in calprotectin more closely resembles the subunit association previously observed in the S100A8 homodimer and provides trans stabilisation for S100A9, which manifests itself in a significantly elongated C-terminal α-helix in the latter. As a consequence, two different putative zinc binding sites emerge at the S100A8/S100A9 subunit interface. One of these corresponds to a high affinity arrangement of three His residues and one Asp side-chain, which is unique to the heterotetramer. This structural feature explains the well known Zn 2+ binding activity of calprotectin, whose overexpression can cause strong dysregulation of zinc homeostasis with severe clinical symptoms.

A novel extracellular EF-hand protein involved in the shell formation of pearl oyster

Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster, Pinctada fucata, according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shell mineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from Escherichia coli. These results suggest that EFCBP might be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation.

G Protein-coupled Receptor Kinase 2-mediated Phosphorylation of Downstream Regulatory Element Antagonist Modulator Regulates Membrane Trafficking of Kv4.2 Potassium Channel

Downstream regulatory element antagonist modulator (DREAM)/potassium channel interacting protein (KChIP3) is a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+-binding proteins with specific roles in different cell compartments. In the nucleus, DREAM acts as a Ca2+-dependent transcriptional repressor, and outside the nucleus DREAM interacts with Kv4 potassium channels, regulating their trafficking to the cell membrane and their gating properties. In this study we characterized the interaction of DREAM with GRK6 and GRK2, members of the G protein-coupled receptor kinase family of proteins, and their phosphorylation of DREAM. Ser-95 was identified as the site phosphorylated by GRK2. This phosphorylation did not modify the repressor activity of DREAM. Mutation of Ser-95 to aspartic acid, however, blocked DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. Treatment with the calcineurin inhibitors FK506 and cyclosporin A also blocked DREAM-mediated Kv4.2 channel trafficking and calcineurin de-phosphorylated GRK2-phosphorylated DREAM in vitro. Our results indicate that these two Ca2+-dependent posttranslational events regulate the activity of DREAM on Kv4.2 channel function.

Allograft inflammatory factor-1 and its immune regulation

The allograft inflammatory factor-1 (AIF-1) is a 17 kDa interferon-γ (IFN-γ) inducible Ca2 + -binding EF-hand protein that is encoded within the HLA class III genomic region. Three proteins including ionized Ca2 + -binding adaptor 1, microglia response factor-1, and daintain are identical with AIF-1. The expression of AIF-1 was mostly limited to the monocyte/macrophage lineage, and augmented by cytokines such as IFN-γ. It was assumed that AIF-1 was a novel molecule involved in inflammatory responses, allograft rejection, as well as the activation and function of macrophages. However, it has been reported that AIF-1 is also expressed in macrophages and microglial cells in autoimmune diseases such as experimental autoimmune encephalomyelitis, neuritis and uveitis models, suggesting that AIF-1 may play a pivotal role in autoimmunity. In the present manuscript, the genomic and functional characteristics of AIF-1 family proteins as well as their immune regulatory effects are reviewed.

Solution Structure of the Cytoplasmic Region of Na+/H+ Exchanger 1 Complexed with Essential Cofactor Calcineurin B Homologous Protein 1

Na+/H+ exchanger 1 (NHE1) regulates intracellular pH, Na+ content, and cell volume. Calcineurin B homologous protein 1 (CHP1) serves as an essential cofactor that facilitates NHE1 exchange activity under physiological conditions by direct binding to the cytoplasmic juxtamembrane region of NHE1. Here we describe the solution structure of the cytoplasmic juxtamembrane region of NHE1 complexed with CHP1. The region of NHE1 forms an amphipathic helix, which is induced by CHP1 binding, and CHP1 possesses a large hydrophobic cleft formed by EF-hand helices. The apolar side of the NHE1 helix participates in extensive hydrophobic interactions with the cleft of CHP1. We suggest that helix formation of the cytoplasmic region of NHE1 by CHP1 is a prerequisite for generating the active form of NHE1. The molecular recognition detailed in this study also provides novel insight into the target binding mechanism of EF-hand proteins.

Implications on zinc binding to S100A2

Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn2+ is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals.

Mouse naked cuticle 2 (mNkd2) as a direct transcriptional target of Hoxc8 in vivo

Mouse naked cuticle 2 (mNkd2), the mammalian homolog of the Drosophila segment polarity gene naked cuticle (nkd), encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. The transcription factor, Hoxc8, a member of the homeobox gene family, is vital for growth and differentiation. Chromatin immunoprecipitation (ChIP) assay, an electrophoretic mobility shift assay (EMSA), and a reporter assay demonstrated that endogenous Hoxc8 protein binds directly to the enhancer region of the mNkd2 gene, implying a Hoxc8-dependent transcriptional activity. Introduction of exogenous Hoxc8 into NIH3T3 cell lines lacking wild-type Hoxc8 dramatically reduced expression of mNkd2 mRNA. If, as the results suggest, mNkd2 is a direct target of Hoxc8, it represents a novel mechanism by which Hoxc8 might cross-talk with the Wnt signaling pathway by regulating mNkd2.

Characterization of a novel EF-hand homologue, CnidEF, in the sea anemone Anthopleura elegantissima

The superfamily of EF-hand proteins is comprised of a large and diverse group of proteins that contain one or more characteristic EF-hand calcium-binding domains. This study describes and characterizes a novel EF-hand cDNA, CnidEF, from the sea anemone Anthopleura elegantissima (Phylum Cnidaria, Class Anthozoa). CnidEF was found to contain two EF-hand motifs near the C-terminus of the deduced amino acid sequence and two regions near the N-terminus that could represent degenerate EF-hand motifs. CnidEF homologues were also identified from two other sea anemone species. A combination of bioinformatic and molecular phylogenetic analyses was used to compare CnidEF to EF-hand proteins in other organisms. The closest homologues identified from these analyses were a luciferin binding protein (LBP) involved in the bioluminescence of the anthozoan Renilla reniformis, and a sarcoplasmic calcium-binding protein (SARC) involved in fluorescence of the annelid worm Nereis diversicolor. Predicted structure and folding analysis revealed a close association with bioluminescent aequorin (AEQ) proteins from the hydrozoan cnidarian Aequorea aequorea. Neighbor-joining analyses grouped CnidEF within the SARC lineage along with AEQ and other cnidarian bioluminescent proteins rather than in the lineage containing calmodulin (CAM) and troponin-C (TNC).

Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin

Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K1=7.1+/-0.4, log K2=4.7+/-0.6, log K3=3.6+/-0.7 and log K4=4.6+/-0.6 in physiological salt buffers) with a [Ca2+](0.5) of approx. 25 microM. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K(d) was determined to be 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.

Kinetoplastid PPEF phosphatases: Dual acylated proteins expressed in the endomembrane system of Leishmania

Bioinformatic analyses have been used to identify potential downstream targets of the essential enzyme N-myristoyl transferase in the TriTryp species, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. These database searches predict ∼60 putative N-myristoylated proteins with high confidence, including both previously characterised and novel molecules. One of the latter is an N-myristoylated protein phosphatase which has high sequence similarity to the Protein Phosphatase with EF-Hand (PPEF) proteins identified in sensory cells of higher eukaryotes. In L. major and T. brucei, the PPEF-like phosphatases are encoded by single-copy genes and are constitutively expressed in all parasite life cycle stages. The N-terminus of LmPPEF is a substrate for N-myristoyl transferase and is also palmitoylated in vivo. The wild type protein has been localised to the endocytic system by immunofluorescence. The catalytic and fused C-terminal domains of the kinetoplastid and other eukaryotic PPEFs share high sequence similarity, but unlike their higher eukaryotic relatives, the C-terminal parasite EF-hand domains are degenerate and do not bind calcium.

EF-hand protein dynamics and evolution of calcium signal transduction: an NMR view

Calcium signaling, one of the most widespread signaling mechanisms in cells, is generally carried out by EF-hand proteins, characterized by a helix-loop-helix motif paired in functional domains. EF-hand proteins may be viewed as molecular switches activated by calcium concentration transients. The EF-hand structural database has grown to a point where meaningful inferences on the functional conformational rearrangements upon calcium binding can be made by comparing a fair number of pairs of end points, i.e., the structures of the apo and calcium-bound forms. More compact descriptors of the movement associated with calcium binding, in terms of principal component analysis of the six interhelical angles, have also become available. Dynamic information obtained by NMR, also with the aid of calcium substitution with paramagnetic lanthanides, is shedding light on the intrinsic amplitude of the conformational degrees of freedom sampled by the various members of the EF-hand superfamily, as well as on the time scales of the motions. Particularly, NMR of lanthanide derivatives helps in capturing long time scale motions. Both static and dynamic pictures reveal a large variety of behaviors. It is increasingly recognized that the EF-hand machinery has differentiated its behavior during evolution in several ways, e.g., by modifying one of the loops, by undergoing a further duplication after the initial motif duplication that originated the functional domain, or by acquiring the ability to dimerize.

Purification and crystallization of the human EF-hand tumour suppressor protein S100A2

S100A2 is a Ca(2+)-binding EF-hand protein that is mainly localized in the nucleus. There, it acts as a tumour suppressor by binding and activating p53. Wild-type S100A2 and a S100A2 variant lacking cysteines have been purified. CD spectroscopy showed that there are no changes in secondary-structure composition. The S100A2 mutant was crystallized in a calcium-free form. The crystals, with dimensions 30 x 30 x 70 microm, diffract to 1.7 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 43.5, b = 57.8, c = 59.8 A, alpha = beta = gamma = 90 degrees. Preliminary analysis of the X-ray data indicates that there are two subunits per asymmetric unit.