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Comprehensive Mutagenesis of the C-Terminal Domain of the M13 Gene-3 Minor Coat Protein: the Requirements for Assembly Into the Bacteriophage Particle

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Comprehensive Mutagenesis of the C-Terminal Domain of the M13 Gene-3 Minor Coat Protein: the Requirements for Assembly Into the Bacteriophage Particle doi:10.1016/S0022-2836(03)00950-1 J. Mol. Biol. (2003) 332, 777–782 COMMUNICATION Comprehensive Mutagenesis of the C-terminal Domain of the M13 Gene-3 Minor Coat Protein: The Requirements for Assembly into the Bacteriophage Particle Gregory A. Weiss1, Tomer A. Roth1, Pierre F. Baldi2 and Sachdev S. Sidhu1* 1Department of Protein Filamentous bacteriophage assemble at the host membrane in a non-lytic Engineering, Genentech Inc., process; the gene-3 minor coat protein (P3) is required for release from 1 DNA Way, South San the membrane and subsequently, for recognition and infection of a new Francisco, CA 94080, USA host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain 2Department of Information (P3-C) that is required for release from the host cell following phage and Computer Science and assembly and contributes to the structural stability of the phage particle. Department of Biological A comprehensive mutational analysis of the 150 residue P3-C revealed Chemistry, Institute for that only 24 side-chains, located within the last 70 residues of sequence, Genomics and Bioinformatics were necessary for efficient incorporation into a wild-type coat. The University of California, 424C results reveal that the requirements for the assembly of P3 into the phage Computer Sciences, Irvine, CA particle are quite lax and involve only a few key side-chains. These find- 92697-3425, USA ings shed light on the functional and structural requirements for filamen- tous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology. q 2003 Elsevier Ltd. All rights reserved. Keywords: phage display; protein engineering; combinatorial mutagenesis; *Corresponding author M13 bacteriophage M13 bacteriophage is a member of the Inoviridae family of viruses, which encapsulate their single- Supplementary data associated with this article can be stranded DNA (ssDNA) genome within a cylindri- found at doi: 10.1016/S0022-2836(03)00950-1 cal, proteinaceous coat.1 Virion length depends on G. Weiss, Department of Chemistry and the Institute the size of the packaged genome, but typically for Genomics and Bioinformatics, University of California, 516 Rowland Hall, Irvine, CA 92697-2025, ranges from 700 nm to 2000 nm; in contrast, the virion has a diameter of only 6–7 nm, hence, the USA; T. Roth, Department of Molecular Pharmacology 2–4 and Biological Chemistry, Northwestern University designation as filamentous bacteriophage. The Medical School, 303 East Chicago Avenue, Chicago, IL filament length is covered by several thousands 60614, USA. copies of the gene-8 major coat protein (P8), while Abbreviations used: ELISA, enzyme-linked the ends are capped by approximately five copies immunosorbant assay; hGH, human growth hormone; each of four minor coat proteins (two different pro- hGHbp, hGH-binding protein; P3, M13 gene-3 minor teins at either end). coat protein; P3-C, C-terminal domain of P3; P3-C1, C- Filamentous bacteriophage are non-lytic and terminal domain 1 of P3; P3-C2, C-terminal domain 2 of assemble by a membrane-associated mechanism.5–7 P3; P8, M13 gene-8 major coat protein; Pn, (where n ¼ 1, 4, or 5), M13 gene-n protein; Pn, (where n ¼ 6, 7, or 9), Coat proteins initially reside as integral inner M13 gene-n minor coat protein; ssDNA, single-stranded membrane proteins with a single transmembrane DNA; wt, wild-type. domain, and assembly occurs at sites formed by E-mail address of the corresponding author: multimers of additional phage-encoded proteins; [email protected] protein-4 (P4) in the outer membrane interacts 0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. 778 Mutagenesis of P3-C with protein-1 and protein-11 (P1 and P11) in the phagemid vector was designed to direct the inner membrane to form an assembly site through secretion of a fusion protein consisting of human which phage are extruded. While in the cytoplasm, growth hormone (hGH) fused to the N terminus the ssDNA is sequestered with another phage- of P3-C. Co-infection of Escherichia coli with the encoded protein (protein-5, P5) which is stripped phagemid and a helper phage resulted in mono- off during assembly. It is believed that assembly is valent phage display of hGH; that is, the pro- initiated when a signature sequence within the duction of phage particles containing ssDNA, the packaging signal, interacts with two predominantly wild-type P3 from the helper different minor coat proteins (P7 and P9) and with phage and no more than one copy of the hGH-P3- P1 at the assembly site. The association of the C fusion. Thus, the level of hGH display served as ssDNA with P7 and P9 forms the leading end of an indicator of the incorporation efficiency, while the phage, which is elongated by extrusion from the excess wt P3 from helper phage supplied the the host and concomitant incorporation of P8. other functions of P3 (i.e. release and stabilization When the end of the ssDNA is reached, assembly of the assembled virion, and host cell recognition is completed by the addition of minor coat proteins and infection). P6 and P3, and the particle is released from the Side-chain contributions to P3-C incorporation host cell membrane. were determined by measuring the effects of trun- Studies of filamentous phage structure and cating mutations. Ala mutations were used biology have shown that both the genome and because this substitution removes side-chain coat are quite malleable, and these findings have atoms past the b-carbon atom, yet it is unlikely to paid major dividends in the development of mol- introduce flexibility into the protein backbone.24 ecular biology methods. Very large inserts can be At positions where Ala was the wild-type (wt) resi- made in the ssDNA genome, as they are accommo- due, Gly substitutions were used to remove even dated readily by a corresponding increase in the the side-chain b-carbon atom. The entire P3-C particle length, and this property has led to the sequence was scanned using an extremely rapid development of versatile cloning vectors.8 The “shotgun scanning” combinatorial mutagenesis coat proteins can tolerate insertions of foreign strategy applied previously to the study of P823 polypeptides, allowing for the development of and several other proteins.25 – 28 Our results offer phage display vectors that permit the display of insights into the relationships between viral struc- polypeptides on the surfaces of phage particles ture and function, and they may provide useful that also contain the encoding DNA.9–11 More guidelines for the design of P3-C variants as recently, phage display methods have been used improved scaffolds for phage display technology. to study the phage itself, in order to better under- stand phage biology and structure, and to further Shotgun scan of P3-C improve phage display technology.12 – 14 Unlike the other phage coat proteins, which are A comprehensive mutational analysis of P3-C small and hydrophobic, mature P3 contains 406 was conducted using ten shotgun scanning residues and consists of several distinct domains libraries within a phagemid vector designed to dis- that together mediate both entry into and release play hGH fused to the N terminus of P3-C.14 Each from the host. Initially, P3 was divided into three library covered nine to 21 contiguous residues and domains separated by glycine-rich linkers: two there was no overlap between the different N-terminal domains (P3-N1 and P3-N2) required libraries. Together, the libraries mutated the entire for host cell infection, and a C-terminal domain sequence of P3-C, with the exception of two (P3-C) required for the release of newly assembled cysteine residues that likely form a disulfide bond virions from the membrane and for structural stab- critical for proper folding. Shotgun scanning ility of the phage particle.15 – 17 However, detailed codons were used to mutate residues to either the analysis of deletion mutants revealed that P3-C wt or Ala in an equimolar ratio (Ala or Gly in contains two functionally distinct subdomains: cases where the wt was Ala), although the redun- C-terminal domain 1 (P3-C1, residues 257–313), dancy of the genetic code necessitated two other which is involved in capping and stabilizing the substitutions at some positions.26 The diversity of assembled phage particle, and C-terminal domain each library was at least tenfold greater than the 2 (P3-C2, residues 314–406), which is required for number of possible amino acid combinations, thus incorporation into the phage coat and subsequent ensuring complete representation of the theoretical release of the assembled virion from the host diversity. membrane.18 While the three-dimensional struc- Phage from each library were cycled through tures of P3-N1 and P3-N2 have been rounds of binding selection with immobilized determined,19 – 22 the structure of P3-C has not. growth hormone-binding protein (hGHbp) as the 29 Here, we map in fine detail the side-chains capture target (Kd ¼ 1.6 nM for binding to hGH), required for the assembly of P3 into the phage as described for a similar analysis of P8.23 Approxi- coat, independent of the other functions of P3. To mately 100 selected clones were sequenced for each facilitate this goal, we used a phagemid-based library, the sequences were aligned, the occurrence phage display system analogous to a system used of wt or each designed mutation was tabulated to study the assembly requirements of P8.23 The at each position, and the wt/mutant ratio was Mutagenesis of P3-C 779 calculated for each mutation at each position.
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