Crystal Structures of NAC Domains of Human Nascent Polypeptide-Associated Complex (NAC) and Its Αnac Subunit

Protein Cell 2010, 1(4): 406–416 Protein & Cell DOI 10.1007/s13238-010-0049-3 RESEARCH ARTICLE Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit ✉ Lanfeng Wang1, Wenchi Zhang1, Lu Wang1, Xuejun C.Zhang1, Xuemei Li1, Zihe Rao1,2,3 1 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China 2 Structure Biology Laboratory, Tsinghua University, Beijing 100084, China 3 Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin 300071, China ✉ Correspondence: [email protected] Received March 28, 2010 Accepted April 12, 2010 ABSTRACT binding with other cytosolic factors (Wiedmann et al., 1994). This complex is widely conserved from archaea to human. Nascent polypeptide associated complex (NAC) and its Some NAC mutants induce early embryonically lethal two isolated subunits, αNAC and βNAC, play important phenotypes in mice, fruit fly, and Caenorhabditis elegans roles in nascent peptide targeting. We determined a 1.9 Å (Deng and Behringer, 1995; Markesich et al., 2000). Experi- resolution crystal structure of the interaction core of NAC ments to determine NAC intracellular localization and heterodimer and a 2.4 Å resolution crystal structure of distribution show that the vast majority of NAC is in the αNAC NAC domain homodimer. These structures provide cytoplasm as a stable heterodimer, and no single subunit and detailed information of NAC heterodimerization and homo-oligomer has been observed under physiologic condi- αNAC homodimerization. We found that the NAC tions (Beatrix et al., 2000). However, drastic variations of domains of αNAC and βNAC share very similar folding relative concentration between the two NAC subunits are despite of their relative low identity of amino acid found in patients of Alzheimer’s disease, Down syndrome, sequences. Furthermore, different electric charge dis- malignant brain tumors, AIDS, and ulcerative colitis (Zuo et tributions of the two subunits at the NAC interface al., 1997; Scheuring et al., 1998; Kroes et al., 2000; Kim et al., provide an explanation to the observation that the 2002), which indicates that imbalanced NAC subunits may heterodimer of NAC complex is more stable than the result in pathological conditions. Sequence analysis predicts single subunit homodimer. In addition, we successfully that the two NAC subunits share low yet recognizable amino built a βNAC NAC domain homodimer model based on acid sequence similarity in a region termed as the NAC homologous modeling, suggesting that NAC domain domain. However, there is a UBA (ubiquitin-associated) dimerization is a general property of the NAC family. domain, only in αNAC but not βNAC, C-terminal to the NAC These 3D structures allow further studies on structure- domain, implicating different functions for the two subunits function relationship of NAC. (Spreter et al., 2005). Saccharomyces cerevisiae genome encodes one αNAC homolog and two βNAC homologs (Shi et KEYWORDS nascent polypeptide-associated complex, al., 1995; Reimann et al., 1999). In contrast, all sequenced αNAC homodimer, βNAC, crystal structure archaebacterial genomes have only one gene, named as aeNAC, homologous to human αNAC (Spreter et al., 2005). INTRODUCTION Therefore, NAC is likely to be an ancient protein complex with some latter-acquired functions. Nascent polypeptide associated complex (NAC) heterodimer At present, it is widely accepted that NAC plays an of αNAC (NACA) and βNAC (BTF3b) is characterized as the important role in cotranslational targeting of nascent poly- first cytosolic factor that binds nascent polypeptides emerging peptides to endoplasm reticulum (ER). Either of the two NAC from the ribosome and prevents the peptides from incorrectly subunits can bind directly with nascent polypeptides 406 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2010 Crystal structures of NAC domain homodimer and heterodimer Protein & Cell emerging from the ribosome, albeit the binding of αNAC is interaction core complex at 1.9 Å resolution and the crystal rather weak. βNAC can directly bind ribosome protein near structure of αNAC NAC domain homodimer at 2.4 Å resolu- the exit site of nascent polypeptide using its N-terminal tion. In addition, our structural analysis and model building ribosome binding motif (Wegrzyn et al., 2006). In contrast, suggested that βNAC subunits can form a homodimer similar αNAC does not bind ribosome protein directly. Nevertheless, to the NAC heterodimer. These structures provide detailed it can interact with ribosome by binding tightly with ribosome information of NAC heterodimerization and an explanation to nucleic acid in a non-sequence-specific manner (Beatrix et why NAC is more stable than an αNAC homodimer. al., 2000). Moreover, the NAC complex can start binding nascent polypeptide when the N terminus of the peptide is RESULTS only 17-residues long from the peptidyl-transferase center, and NAC starts being released after the polypeptide becomes Structure of human NAC complex longer than 30 residues. Protease digestion assays confirm the NAC protection for nascent polypeptide (Wang et al., We coexpressed and copurified the full length recombinant 1995). The binding and releasing of NAC for nascent proteins of human αNAC (GenBank: AAX14393.1, residues polypeptide appear in quantal units instead of a residue-by- 1–215) and βNAC (GenBank: AAH08062.1, residues 1–162) residue manner. This binding is independent of signal peptide from E. coli. Solution study showed that αNAC and βNAC sequences, and yet NAC cooperates with signal recognition recombinant proteins formed a stable heterodimer ( Supple- particle (SRP) for correctly targeting and folding of nascent mental Fig. 1A). NAC crystals of good diffraction quality polypeptide in the cotranslational process (Lauring et al., appeared six months after setting up the crystallization drops. 1995a, b; Powers and Walter, 1996; Moller et al., 1998b). The reservoir solution was 0.1 M sodium citrate (pH 5.6), However, yeast NAC homolog (termed as EGD complex) is 0.1 M NaCl, and 12% (w/v) polyethylene glycol (PEG) 4000. not prerequisite for growth (Reimann et al., 1999), but is Regarding the prolonged crystallization time, we speculated necessary for protein targeting to mitochondria (George et al., that the protein sample was processed by some incidentally 1998; Fünfschilling and Rospert, 1999). In addition, in yeast contaminated protease(s), which promoted the crystallization. both subunits including their NAC domains have been shown Phases of the crystal structure were solved at 1.9 Å resolution to be ubiquitinated in a lysine dependent manner (Panasenko using the single wavelength anomalous diffraction (SAD) data et al., 2006, 2009). Besides functioning in cytosol, NAC can collected from a potassium iodine derivative. Subsequently, it tightly bind in nucleus with X-junction in DNA replication forks was found that the crystal structure contained only the NAC and homolog recombinant regions (Whitby and Dixon, 2001). domains from both αNAC (residues 79–132, designated as Therefore, the heterodimer of NAC appears to be multi- NACα) and βNAC (residues 53–110, named as NACβ) forming functional. a heterodimer of dimensions of 30 × 34 × 45 Å (Fig. 1A). The In the absence of a hetero-partner, for example when solvent content of the crystal form was estimated to be 63% 3 overexpression or downregulation of a single subunit, the (VM = 3.3 Å /Da) with one such heterodimer per asymmetric remaining dominant subunit may have functions independent unit. The crystal structure was refined to R factor of 0.196 of nascent peptide binding (Thiede et al., 2001; Spreter et al., (and R-free of 0.245) with excellent geometry (Table 1). Taken 2005). For instance, isolated αNAC is a multiple functional together, we obtained the crystal structure of a stable core of protein taking part in transcription regulation (Moreau et al., the human NAC heterodimer. 1998; St-Arnaud, 1998; St-Arnaud and Quelo, 1998; Yotov In this complex, NACα and NACβ subunits shared a similar et al., 1998), cell proliferation, and differentiation (Al-Shanti V-shaped structure. The topology of the heterodimer is shown et al., 2004; Lopez et al., 2005; Al-Shanti and Aldahoodi, in Fig. 1B. NACα consisted of helix α1 and strands β1–β6; and 2006). Similarly, isolated βNAC (also called BTF3b) was NACβ consisted of strands β1’–β6’, helices α1’ and α2’ initially identified as an alternative splicing isoform of the basic (structural elements of NACβ are denoted as primes here- transcription factor (BTF3a). However, βNAC is transcription- after). In NACα, α1, β1, β4, and β5 were arranged in one arm ally inactive although it has the same ability as BTF3a to bind of the V-shaped structure, and β2, β3, and β6 were arranged RNA polymerase II (Zheng et al., 1987, 1990). Structural in the other arm. Similarly, in NACβ, β1’, β4’, and β5¢ were basis of such distinct functions remains to be determined. assembled in one arm, and β2’, β3¢, β6’, α1’, and α2’ were in The crystal structure of aeNAC shows that it forms a the other arm. The two V-shaped structures packed with each homodimer via an interaction between the NAC domains other in a head-to-head manner to generate a 12-stranded β- (Spreter et al., 2005). However, until now there has been no barrel-like heterodimer (NACα-NACβ) with six strands in each experimental data on how human NAC heterodimer is made of the two major β-sheets (Fig. 1A). Structure based from αNAC and βNAC and what the structural difference is sequence alignment (Fig. 1C) showed that there is neither between NAC and its subunit homodimer. In this study we insertion nor deletion between the NAC domains of αNAC used the crystallography method to address these questions. and βNAC subunits. Although amino acid-sequence identity We determined the crystal structure of NAC hetero-dimeric between the NAC domains of NACα and NACβ is low (10/48 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2010 407 Figure 1.

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