SRP

Signal Recognition Particle

GTP-binding proteins necessary for proper export/transport of secretory and plasma membrane proteins

Prokaryotic: composed of 2 proteins and an RNA, the SRP and the receptor protein act as GAPs (guanine nucleotide activating proteins) for each other

Ffh Fifty-four homologue (homologous to SRP54)

• SRP

• also known as P48

• binds to ~50 nucleotide stem (domain IV) in the 4.5 S RNA, it is bound on one end by an tetraloop and contians two short internal loops (symmetric and asymmetic loops) with highly conserved sequences

• 453 amino acids (E. coli)

• composed of N-terminal domain (N) with 4 helices, middle GTPase domain (G), and a C-terminal M domain (methionine-rich, binds signal sequence); the M domain is linked to the NG domains by a long, slender linker as determined by biochemical, genetic, and scanning transmission electron microscopy, also denoted membrane-attachment site on ribosomes

• From the GDP-Mg²⁺ bound form of the Thermus aquaticus Ffh (Walter's group), the GTP binding site can be hypothesized using Rasmol to define residues within 5 of the GDP. These are: Lys-246, Thr-245, Gly-271, Ser-273, Val-272, Glu-274, Thr-113, Thr-112, Lys-111, Gly-110, Ser-109, Gly-108, and Gln-107.

• According to Freymann's paper on his new, yet unreleased structures, Mg²⁺ is (hypothesized) to be coordinated by Thr-112 OG1 and a b-phosphate oxygen in the GMPPNP model as there is no Mg²⁺ present in the structure. Leu-106, Leu 192, and Gln-107 of the P loop are involved in nucleotide binding as are Asp-248, Lys-111, Thr-113, Arg-191, and Asp-187.

FtsY

• SRP receptor

• homologue to eukaryotic SRa

• may bind to membrane protein corresponding to eukaryotic SR? (otherwise no SR? homologue in prokaryotes)

• 497 amino acids (E. Coli)

• composed of a C-terminal G domain, a GTPase domain (G) that binds and hydrolyzes the GTP, an N domain that has four-helices like the N domain in Ffh, and the acidic N-terminal A domain that associates with the membrane lipids

• found as a peripheral membrane protein but most often found in the cytoplasm of the cell

• interacts with anionic phospholipids in the membrane, not a protein

• The residues in FtsY homologous to those in the GTP-binding site of Ras have been described. These are 5-10 away from a docked (but not yet minimized) GDP-MG²⁺ in the apo-FtsY but may be involved in the GTP binding site. These are: Lys-205, Thr-206, Asp-330, Thr-331, Phe-332, Arg-333, Gly-367, Ala-358, Gly-385, Thr-446, Asp-449, Gly-450, and Thr-451.

• 4.5 S RNA

• also called Ffs

• 114 nucleotides

• homologous to stem VIII to the 7 S RNA in eukaryotes

• also binds EF-G so it is involved in translation, both Ffh and EF-G bind to conserved helix 8 but to different nucleotides

Eukaryotic: SRP is composed of 6 proteins and an RNA, the receptor is composed of two proteins

SRP Proteins: SRP9, SRP14, SRP19, SRP54, SRP68, SRP72

• SRP M domain has been crystallized (while this has not been in bacteria) and consists of 7 ?-helices. It has a groove in helix 1 and a hydrophobic groove made up of Met351, Ile352, Pro353, Gly354, Phe355 and Phe359 that could be the site of the signal peptide recognition. It binds the RNA at helices 5, 6, and in parts of helices 4 and 7.

• 7 S RNA

• SRP54 complexes with domain VII of the 7 S RNA and binds the signal sequence

• Alu domain (confers elongation arrest activity) is composed of SRP9/SRP14 and the Alu sequences of the 7 S RNA

• SRP68 and SRP72 form a complexand bind in the S-domain of the 7S RNA

• SRP19 binds at the helix 6 of the 7S RNA

• SRP54 binds at the helix 8 of the 7S RNA

• Receptor Proteins: SRa (docking protein, 69-72 kDa) and SRb (transmembrane protein, 30 kDa)

Possible points for further study (remaining questions)

• How does the binding of GTP lead to a conformational change in the GTPase of the SRP or its receptor?

• What is the structure of the SRP:SRP receptor (SR) complex (both bound GTP)? Why does this form when GTP is bound but not when GDP is bound?

• How does binding to the lipid bilayer/membrane affect the conformation of the SRP GTPase and lead to GTP hydrolysis?

• How does the Ffh M-domain recognize and bind the signal sequence?

Structural data in the Protein Data Bank

1FTS.pdb

• SRP receptor from E. coli

• expressed N and GTPase domain residues 197-497, residues 201-495 included in the structure

• solved by x-ray crystallography to 2.2 resolution

• apo, no SO₄

• Montoya, G., Svensson, C., Luirink, J., Sinning, I. Expression, crystallization and preliminary X-ray diffraction study of FtsY, the docking protein of the signal recognition particle of E. coli. (1997) Proteins 28: 285-288; Montoya, G., Svensson, C., Luirink, J., Sinning, I. Crystal structure of the NG domain from the signal-recognition particle receptor FtsY. (1997) Nature 385:365-368.

1FFH.pdb

• SRP from Thermus aquaticus

• N and GTPase domains expressed in E. coli, residues 2-295 included in the structure

• solved by x-ray crystallography to 2.05 resolution

• 1 hydrated Mg, 124 crystallographic water molecules

• Freymann, D. M., Keenan, R. J., Stroud, R. M., Walter, P. Structure of the conserved GTPase domain of the signal recognition particle. (1997) Nature 385:361-364.

1DUH.pdb

• conserved domain IV of Escherichia Coli 4.5S RNA

• solved by x-ray crystallography to 2.70 resolution

• nucleotides 31-74

• with PGP (Guanosine-5',3'-Diphosphate), Lu, Mg, SO₄, 6 water molecules

• Jovine, L., Hainzl, T., Oubridge, C., Scott, W. G., Li, J., Sixma, T. K., Wonacott, A., Skarzynski, T., Nagai, K. Crystal Structure of the Ffh and EF-G Binding Sites in the Conserved Domain IV of Escherichia Coli 4.5S RNA. (2000) Structure Folding and Design (London) 8: 527-540.

1HQ1.pdb

• Ffh from E. coli with 4.5 S RNA domain IV

• C-terminal domain (residues 328-432) with nucleotides 32-74 of the RNA

• solved by x-ray crystallography to 1.52 resolution

• 4 Mg, 3 K, 287 crystallographic water molecules

• Batey, R. T., Sagar, M. B., Doudna, J. A. Structural and Energetic Analysis of RNA Recognition by a Universally Conserved Protein from the Signal Recognition Particle. (2001) J.Mol.Biol. 307: 229-246.

1J8M.pdb

• SRP54 from the Archaeon Acidianus Ambivalens

• GTPase domain, residues 3-297

• solved by x-ray crystallography to 2.00 resolution

• 178 crystallographic water molecules

• Montoya, G., Te Kaat, K., Moll, R., Schafer, G., Sinning, I.: The Crystal Structure of the Conserved GTPase of SRP54 from the Archaeon Acidianus Ambivalens and its Comparison with Related Structures Suggests a Model for the SRP-SRP Receptor Complex. (2000) StructureFolding and Design 8:515-525.

1J8Y.pdb

• T112A mutant of 1J8M.pdb: SRP54 from the Archaeon Acidianus Ambivalens

• GTPase domain, residues 3-297

• solved by x-ray crystallography to 2.00 resolution

• Montoya, G., Te Kaat, K., Moll, R., Schafer, G., Sinning, I.: The Crystal Structure of the Conserved GTPase of SRP54 from the Archaeon Acidianus Ambivalens and its Comparison with Related Structures Suggests a Model for the SRP-SRP Receptor Complex. (2000) StructureFolding and Design 8:515-525.

1NG1.pdb (WITH GDP and Mg)

• N and GTPase domains of Ffh from Thermus Aquaticus

• residues 1-294

• solved by x-ray crystallography to 2.03 resolution

• 4 ACY (acetic acid), 2 ENDO (1 of each: 1,2-ethanediol, ethylene glycol), 1 GDP (guanosine-5'-diphosphate), 2 hydrated Mg, 3 Cd, 201 crystallographic water molecules

• Freymann, D. M., Keenan, R. J., Stroud, R. M., Walter, P. Functional Changes in the Structure of the SRP GTPase on Binding GDP and Mg²⁺GDP. (1999) Nat. Struct. Biol. 6:793-801.

2NG1.pdb (GDP but NO Mg)

• N and GTPase domains of Ffh from Thermus Aquaticus

• residues 2-294

• solved by x-ray crystallography to 2.02 resolution

• 2 ENDO (1 of each: 1,2-ethanediol, ethylene glycol), 1 DOX (dioane), 1 GDP, 72 crystallographic water molecules

• Freymann, D. M., Keenan, R. J., Stroud, R. M., Walter, P. Functional Changes in the Structure of the SRP GTPase on Binding GDP and Mg²⁺GDP. (1999) Nat. Struct. Biol. 6:793-801.

3NG1.pdb (NO Mg or GDP) (BUT NOT TRUE APO AS THERE ARE METAL + SO₄)

• N and GTPase domains of Ffh from Thermus Aquaticus

• residues 1-294 and residues 1-294 (dimer)

• solved by x-ray crystallography to 2.30 resolution

• 2 ENDO (1 of each for each dimer: 1,2-ethanediol, ethylene glycol), 2 hydrated Mg, 3 Cd (for each dimer), SO₄ (for each dimer), 113 crystallographic water molecules (for each dimer)

• Freymann, D. M., Keenan, R. J., Stroud, R. M., Walter, P. Functional Changes in the Structure of the SRP GTPase on Binding GDP and Mg²⁺GDP. (1999) Nat. Struct. Biol. 6:793-801.

1QB2.pdb

• SRP54 M domain (signal peptide binding) from Homo sapiens

• M domain, residues 326-432 residues 326-434 (dimer)

• solved by x-ray crystallography to 2.10 resolution

• 91 crystallographic water molecules

• W. M. Clemons Jr., Gowda, K., Black, S. D., Zwieb, C., Ramakrishnan, V. Crystal Structure of the Conserved Subdomain of Human Protein SRP54M at 2.1 Angstroms Resolution: Evidence for the Mechanism of Signal Peptide Binding. (1999) J.Mol.Biol. 292:697-705.

1D4R.pdb

• 29-Mer Fragment of SRP RNA Helix 6 from Homo sapiens

• includes nucleotides 1-24 2-29 1-29

• solved by x-ray crystallography to 2.00 resolution

• 1 A23 (adenosine-5'-phosphate-2',3'-cyclic phosphate), 2 GDP (guanosine-5'-diphosphate), 5 Mg, 100 crystallographic water molecules

• Wild, K., Weichenrieder, O., Leonard, G. A., Cusack, S. The 2 Structure of Helix 6 of the Human Signal Recognition Particle RNA. (1999) Structure Folding and Design (London) 7:1345-1352.

1E8O.pdb

• Core of the Alu 5' domain of the human SRP: SRP9 SRP14 (truncated after Lys-107)

• G1-U47 Of 7Sl RNA Plus A 5'Gg and A 3'C: 7SL RNA, 5'-R(GdpGpGpCpCpGpGpGpCpGpCpGpGpUpGpGpCpGpCpGpCpGpCpCpUpGpUpApGpUpCpCp CpApGpCpUpApCpUpCpGpGpGpApGpGpCpUpC)-3'

• includes residues 2-75 5-75 of SRP9, 2-95 2-95 of SRP14, 49 nucleotides of 7SL RNA

• solved by x-ray crystallography to 3.20 resolution

• 3 SO₄, GDP, 22 crystallographic water molecules

• Weichenrieder, O., Wild, K., Strub, K., Cusack, S. Structure and Assembly of the Alu Domain of the Mammalian Signal Recognition Particle. (2000) Nature. 408:167-173.

1E8S.pdb

• Alu domain of the mammalian SRP (potential Alu retroposition intermediate)

• SRP9, SRP14, 7Sl RNA, 5'-(GpCpUpApGpCpGpApGpApCpCpCCpGpUpCpUpCpUpGpCpCpGpGpGpCp

• GpCpGpGpUpGpGpCpGpCpGpCpGpCpCpUpGpUpApGpUpCpCpCpApGpCpUpApCpUpCpGpGpGpApGpGpCpUpGpApGpGpUpG GpGpApGpGpApUpCpGpCpUpApGpUpC)-3'

• G1-U64 and A283-U299 Of 7SL RNA, circular permutation (G1 Linked To U299), additional nucleotides: 5' G280, C281, U282 and 3' A65, G66, U67, C68

• includes residues 5-75 of SRP9, 2-95 of SRP14, nucleotides of 7SL RNA and added piece 380-389, 101-166

• solved by x-ray crystallography to 4.00 resolution

• 2 Eu

• Weichenrieder, O., Wild, K., Strub, K., Cusack, S. Structure and Assembly of the Alu Domain of the Mammalian Signal Recognition Particle. (2000) Nature. 408:167-173.

1CQ5.pdb

• SRP RNA Domain IV from E. Coli

• nucleotides 1-43

• solved by NMR spectroscopy (average structure, all ten structures available in 1CQL.pdb)

• Schmitz, U., Behrens, S., Freymann, D. M., Keenan, R. J., Lukavsky, P., Walter, P., James, T. L. Structure of the Phylogenetically Most Conserved Domain of SRP RNA. (1999) RNA. 5:1419-1429.

1JID.pdb

• Human SRP19 In Complex With Helix 6 Of Human SRP RNA

• solved by x-ray crystallography to 1.80 resolution

• includes residues 5-118 of human SRP19 and nucleotides 135-162 of the helix 6 of human SRP RNA

• 2 Mg, 271 crystallographic water molecules, 1 ADENOSINE-5'-PHOSPHATE-2',3'-CYCLIC PHOSPHATE, 1 BRO (BROMO GROUP)

• Wild, K., Sinning, I., Cusack, S. Crystal Structure of an Early Protein-RNA Assembly Complex of the Signal Recognition Particle. (2001) Science. 294:598-601.

1914.pdb

• SRP Alu RNA Binding Heterodimer SRP9/14

• Functional and active in translation arrest and release of translation arrest

• solved by x-ray crystallography to 2.53 resolution

• total residues included: 185

• from Mus Musculus

• BME (BETA-MERCAPTOETHANOL), PO_4, 16 crystallographic water molecules

• Birse, D. E., Kapp, U., Strub, K., Cusack, S., Aberg, A. The crystal structure of the signal recognition particle Alu RNA binding heterodimer, SRP9/14. (1997) EMBO J. 16:3757-3766.

2FFH.pdb

• Ffh from Thermus aquaticus

• residues 1-425 (three of 1-418)

• solved by x-ray crystallography to 3.20 resolution

• Cd, SO_{4 ,} (1 for each fragment)

• Mutation Ala48Thr

• Keenan, R. J., Freymann, D. M., Walter, P., Stroud, R. M. Crystal Structure of the Signal Sequence Binding Subunit of the Signal Recognition Particle. (1998) Cell(Cambridge, Mass.) 94:181-191.

Structural data we should obtain

1JPJ.pdb (Release Feb. 2002) N1

• GMPPNP Complex of SRP GTPase NG Domain

• solved by x-ray crystallography to 2.3 resolution

• Padmanabhan, S., Freymann, D.M. (Northwestern) (The conformation of bound GMPPNP suggests a mechanism for gating the active site of the SRP GTPase. (2001) Structure with Folding and Design 9:859-867)

1JPN.pdb (Release Feb. 2002) N2

• GMPPNP Complex of SRP GTPase NG Domain (closing loop here is slightly more open)

• solved by x-ray crystallography to 1.9 resolution

• Padmanabhan, S., Freymann, D.M. (Northwestern) (The conformation of bound GMPPNP suggests a mechanism for gating the active site of the SRP GTPase. (2001) Structure with Folding and Design 9:859-867)

coordinates for SRP:SR complex (proposed by I. Sinning)

• model of A. ambivalens Ffh complexed with E. coli FtsY using the nitrogenase iron protein (1NIPA and 1N2C) (D.C. Rees)

• this was a superimposition

• Ref: Montoya, G., te Kaat, K., Moll, R., Schaefer, G., Sinning, I. The crystal structure of the conserved GTPase of SRP54 from the archaeon Acidianus ambivalens and its comparison with related structures suggests a model for the SRP-SRP receptor complex. (2000) Structure 8:515-525.