1. J Proteins Catalytically Activate Hsp70 Molecules to Trap a Wide Range of Peptide Sequences 
Benjamin Misselwitz, Oliver Staeck, Tom A Rapoport 
Molecular Cell 
Volume 2, Issue 5, Pages (November 1998)
DOI: /S (00)

2. Figure 1 BiP Binds to an Immobilized Hydrophobic Peptide
(A) Peptide P15 (ALLLSAPRRGAGKKC) was immobilized on the surface of an SPR chip via its C-terminal cysteine. At zero time (arrow), BiP (20 μM) was added to a buffer solution passed continuously over the chip. The experiments were performed in the presence of 2 mM ATP or ADP. The increase in RU is proportional to the mass of BiP bound to the surface. At the time point where the curves begin to fall (arrow), the BiP solution was replaced by buffer. The association kinetics did not conform to a simple bimolecular reaction, whereas the dissociation kinetics could be fitted with a single exponential (not shown). BG (dashed curve), background binding to a chip lacking P15.
(B) As in (A), except that 10 μM solutions of wild-type (wt) BiP or of truncation mutants (dashed curves) were passed over the chip. BiPΔlid lacks the C-terminal lid domain, and the ATPase domain mutant lacks both the lid and the peptide-binding domains.
Molecular Cell 1998 2, DOI: ( /S (00) )

3. Figure 2 The J Domain of Sec63p Stimulates ATP Hydrolysis by BiP
(A) The peptide P15 was immobilized on an SPR chip and, at the zero time point, a buffer solution passing over it was replaced by a 10 μM BiP solution containing 2 mM ATP or ADP and the indicated concentrations of purified J domain (J). Where the curves start to fall, the solutions were changed back to buffer.
(B) A single-turnover ATPase assay was performed at 0°C by mixing 980 pmol BiP with 50 pmol γ-32P-ATP. At the zero time point, either buffer (closed circles) or 2.5 nmol J domain (closed triangles) were added. In the experiment shown in open symbols, 2 mM unlabeled ATP was added to the solutions to prevent rebinding of dissociated labeled nucleotide. The amount of 32P-phosphate generated with time was determined by thin layer chromatography and expressed as percentage of the total radioactivity.
Molecular Cell 1998 2, DOI: ( /S (00) )

4. Figure 3 J-Activated Peptide Binding by BiP
(A) The peptide P15 and purified J domain were coimmobilized on the same SPR chip by coupling via their single cysteines (approximately 1000 RU P15 and 100 U J domain). At the zero time point, a buffer solution passing over the surface was replaced by a 15 μM BiP solution containing either 2 mM ATP or ADP. Where the curves start to fall, the solution was changed back to buffer. In controls (dashed curves), the binding of BiP was tested with chips containing the same amounts of immobilized J domain (J alone ATP) or peptide (P15 alone ADP, ATP).
(B) As in (A), except that the experiment was carried out with a BiP mutant defective in ATP hydrolysis, carrying a point mutation in the ATPase domain (hydrolysis mutant).
(C) As in (A), except that BiP binding was tested with a chip that contained the defective J domain of the sec63-1 mutant instead of the wild-type J domain coimmobilized with P15.
(D) SPR chips were generated that contained either immobilized P15 alone (450 RU) or the same amount of P15 and increasing amounts of a shortened version of the J domain (J). The numbers in parentheses give the relative amounts of immobilized J domain (1 corresponds to 35 RU and to a molar ratio of peptide P15 to J domain of approximately 60 to 1). Solutions containing ATP and BiP at different concentrations were passed over the chips and the response followed over time. The maximum RU were plotted versus the BiP concentration (solid curves). The same experiment was carried out with ADP (dashed curves). These curves are superimposable regardless of the amounts of immobilized J domain. Maximum binding of BiP in the presence of ATP to a chip containing the highest amount of J domain alone was 850 RU.
Molecular Cell 1998 2, DOI: ( /S (00) )

5. Figure 4
BiP Is Bound to Peptide in Its ADP Form and Released after Nucleotide Exchange
(A) An SPR chip was generated that contained both immobilized peptide P15 and J domain. At the zero time point, a buffer passing over the chip was replaced by 5 μM BiP solutions containing 2 mM total nucleotide (ATP plus ADP) but a variable percentage of ATP (thus, 0% and 100% indicate the exclusive presence of ADP and ATP, respectively). Note that a decrease in the share of ATP initially increases the level of BiP binding, indicating that ATP replaces ADP before BiP dissociates from the peptide.
(B) BiP was prebound to SPR chips, either in the presence of ADP to one containing immobilized peptide P15 alone (P15; solid curves) or in the presence of ATP to one containing both P15 and coimmobilized J domain (P15 + J; broken curves). The dissociation of BiP from the chips was followed in the presence of 2 mM ATP or ADP, as indicated, in the presence of 2 mM competitor peptide to prevent rebinding of BiP to the surface. The starting points for the dissociation experiments were adjusted to be equal, which was possible because in each case the dissociation kinetics was monophasic (it could be fitted by a single exponential; not shown). The solid and broken curves are superimposable, indicating that after J activation, BiP is bound to P15 in its ADP form.
Molecular Cell 1998 2, DOI: ( /S (00) )

6. Figure 5
The J Domain Activates BiP to Bind to Peptides that It Would Not Bind on Its Own
(A) BiP at different concentrations in either ATP or ADP was passed over SPR chips that contained either peptide P15 alone (open symbols on broken curves) or both immobilized P15 and J domain (closed symbols on solid curves). The response was followed with time and the maximum response plotted versus the BiP concentration. Binding to a chip containing only the J domain was determined for each BiP concentration and subtracted from the values obtained with J domain plus peptide. The largest values subtracted at the highest BiP concentration were 600–700 RU for ATP and 330–350 RU for ADP.
(B–E) As in (A), except that the hydrophilic peptides NB1–NB4 were coimmobilized with the J domain. The binding with peptide alone (broken curves) was low in both ADP and ATP, and the curves were almost superimposable.
(F and G) As in (A), except that hen egg lysozyme or bovine heart cytochrome c was coimmobilized with the J domain. The binding with peptide alone (broken curves) was low in both ADP and ATP and the curves were almost superimposable.
Molecular Cell 1998 2, DOI: ( /S (00) )

7. Figure 6
Models for the Coupling of the Cycles of Peptide Binding and ATP Hydrolysis of BiP
BiP is initially in the ATP form and ends up bound to peptide in its ADP form. Two possible pathways for the conversion of one into the other are indicated. In one route, BiP-ATP interacts with the J domain (J) to generate an activated state that either contains ATP or ADP (BiP-ATP* or BiP-ADP*); this species is rapidly converted into the ADP form with a closed peptide-binding pocket, trapping peptides that are encountered during its lifetime. In the other pathway, BiP-ATP binds peptide first, the complex then interacts with the J domain, and the peptide is trapped in the closed pocket. In either case, dissociation of peptide from BiP-ADP occurs after nucleotide exchange (diagonal arrow).
Molecular Cell 1998 2, DOI: ( /S (00) )