We sought to develop a simple isobaric CLMS-based strategy, called Qlinker, that permits probing conformational changes in proteins/protein complexes by reliably quantifying the relative abundances of crosslinker-modified peptides derived from samples of the protein/complex in different structural states. An important consideration when designing isobaric amine-reactive crosslinkers is that the m/z’s of the reporter ions do not overlap the m/z’s of other fragment ions, which could skew quantification. Unlike iTRAQ or TMT modified peptides, which cannot generate unmodified b1⁺ ions (because all peptide N-termini are labeled), the Qlinker-modified peptides, which are generated by enzymatic digestion after the crosslinking reaction is complete, have free N-terminal amines and will generate unmodified b1⁺ ions. (A b1⁺ ion is an ion with a charge state of +1 corresponding to the first N-terminal amino acid residue after breakage of the first peptide bond.) Since isoleucine and leucine residues generate b1⁺ ions at 114.09 m/z and asparagine and aspartate residues generate b1⁺ ions at 115.05 m/z and 116.03 m/z, respectively, we sought to incorporate a moiety into the Qlinker that generates reporter ions with m/z’s that do not overlap with the m/z’s of b1⁺ and immonium ions. Thus, we decided to use 1-imino-2.6-dimethylpiperidin-1-ium as the reporter ion moiety, which is the same as the moiety used in 126–134 TMT reagents (Figure 1—figure supplement 1). We searched 83,242 mass spectra obtained from the analysis of a BS3 crosslinked sample of the 1 MDa yeast Mediator complex and only found 141 spectra (0.17%) containing 126.128 ions and 18 spectra (0.02%) containing 127.131 ions within a 40 ppm mass tolerance. These results indicated that the 126.128 and 127.131 reporter ions can be used for accurate quantification of Qlinker-modified peptides with little interference from fragment ions derived from peptides that are not modified with Qlinkers.

Another important consideration in the design of the amine-reactive Qlinker is that it can react with ɛ-amines of lysine that are in close proximity. Iminodiacetic acid, iminodipropionic acid, and iminodibutyric acid were considered as base structures. Due to the flexibility of lysine side chains, it was found that Cα-Cα distances of residues crosslinked with amine-reactive crosslinkers are more useful as distance restraints than the distances between crosslinked ɛ-amines. The BS3 crosslinker has a spacer arm of 11.4 Å when fully extended and can crosslink lysine residues whose Cα atoms are up to 30 Å apart (Merkley et al., 2014). The zero-length crosslinker EDC/DMTMM can crosslink a lysine residue and an aspartate or a glutamate residue with Cα atoms up to 20 Å apart (Leitner et al., 2014). We reasoned that crosslinkers based on iminodiacetic acid or iminodipropionic acid with extended crosslinker spacer arms of 7.6 Å and 10.1 Å, respectively, would provide similar structural information, while the backbone of an iminodiacetic acid-based reagent would be more restricted than that of an iminopropionic acid-based reagent. At the same time, iminodibutyric acid quickly generates a dark red color during activation (probably due to lactone formation). We decided to use iminodipropionic acid as the base structure for the new quantitative Qlinker (Figure 1—figure supplement 1).

The two isobaric Q2linkers are named C1q2 and C2q2, synthesized from 1-¹³C and 2-¹³C bromoacetic acid (BAA) respectively (Figure 1a, Figure 1—figure supplement 1, and ‘Materials and methods’). A typical experiment uses equal amounts of C1q2 and C2q2 to crosslink equal amounts of proteins or protein complexes in different structural states. After quenching the reactions with ammonium sulfate, the two samples are combined and mixed well, reduced and denatured, trypsin digested, and analyzed by MS using a stepped HCD MS2 method (Diedrich et al., 2013). The Qlinker-modified peptide spectra are identified as either monolinks or crosslinks by different database search engines. The ion signals from the 126.1277 (C1q2) and 127.1311 (C2q2) reporter ions are then extracted from identified spectra to compute the relative abundance of the modified peptides in the different samples (Figure 1a).

To evaluate the ability of Q2linkers to provide accurate quantification of crosslinker-modified peptides, we crosslinked equal amounts of affinity-purified RNA polymerase I (pol I) with either C1q2 or C2q2, digested the two samples separately, and then combined the resulting peptides at known ratios for MS analysis (Figure 1—figure supplement 2). Examples of a monolinked peptide spectrum (Figure 1b) and a crosslinked spectrum (Figure 1c) are shown, with the reporter ion intensities from the indicated mixing ratios. The 126 and 127 reporter ion intensities were extracted from the identified spectra corresponding to 298 monolinks and 32 crosslinks, within a mass tolerance of 0.005 Da (~40 ppm) and adjusted by correction factors calculated from the isotopic distribution (‘Materials and methods’). The measured log2 ratios of 127/126 intensity were then compared with the expected log2 ratios. The measured ratios from both monolink spectra (Figure 1d) and crosslink spectra (Figure 1e) were strongly correlated with the expected ratios with slopes close to 1 (Figure 1—figure supplement 3). At each mixing ratio, the average measured ratio was slightly lower than the expected ratio (intercept ~–0.4), with a standard deviation around ±0.25, suggesting that there were slightly more crosslinker-modified peptides in the C1q2 sample prior to mixing. This may be due to some peptide loss in the C2q2 sample during trypsin digestion and C18 preparation prior to mixing, or there was slightly less C2q2 crosslinker in the reaction. This also highlights the importance of combining crosslinked samples at an early stage to minimize sample handling variations. The standard deviation is larger for the samples mixed at 5:1 and 10:1 C1q2 labeled (126) to C2q2 labeled (127) (around ±0.42 and ±0.78, respectively). We think this could be due to either the higher natural isotope contribution (~10%) from the 126 reporter ion to the 127 reporter ion, or interference from other fragment ions. Overall, these experiments demonstrate that the Q2 quantification strategy can accurately reflect the relative abundance of Q2linker-modified peptides.

We then affinity-purified pol II from a yeast strain expressing a FLAG-tagged version of pol II subunit Rpb3 and crosslinked equal amounts of the 12 subunit pol II complex with Q2linkers in the presence and absence of α-amanitin. No major structural rearrangements occur in pol II upon α-amanitin binding (1i3q.pdb for free pol II and 1k83.pdb and 3cqz.pdb for α-amanitin bound pol II). The distributions of log2(127/126) ratios for monolinked (total 174), intralinked (total 154), and interlinked (total 77) peptides were all centered around 0 with no significant differences. Also, 98% of the log2 ratios fell within the range ±0.5 (Figure 1—figure supplement 4). This experiment demonstrates that Q2linkers provide reliable crosslink and monolink quantification with no abnormal ratios when applied to a protein complex in a situation where no significant conformational changes and/or structural rearrangements are known to occur. We initially designed this experiment thinking that α-amanitin binding to pol II might affect Q2linker modification of a lysine residue(s) near the α-amanitin binding site, and we might be able to identify the α-amanitin binding site by analysis of quantitative Q2linker MS data. Unfortunately, no Q2linker-modified peptides were identified near the site where α-amanitin binds. This experiment also highlights one of the limitations of residue-specific, quantitative CLMS methods in general. Reactive residues must be available near the region of interest, and the modified peptides must be identifiable by MS.

Protein biosensors are polypeptides that undergo conformational changes or switches upon receiving an input signal. We next designed experiments to see whether Q2linkers can detect conformational changes in biosensors. We tested two commercially available proteins: maltose binding protein (MBP) and calmodulin (CaM). MBP is a 370 amino acid polypeptide that buries the maltose ligand between a cleft formed by its two domains and exhibits a conformational change from an open ligand-free conformation (1mpb.pdb) and a closed maltose-bound conformation (1n3w.pdb). However, it is the ‘balancing interface’ on the opposite side of the ligand binding cleft that maintains the open conformation; upon maltose binding, this interface is disrupted and becomes more solvent exposed (Telmer and Shilton, 2003). To evaluate the ability of Q2linkers to detect conformational changes in MBP, we performed a crosslinker swapping experiment in which we crosslinked 10 ug of MBP in the presence and absence of 10 mM maltose with C1q2 and C2q2, respectively, in one experiment, and, in a second experiment, we crosslinked MBP in the presence and absence of maltose with C2q2 and C1q2, respectively. In each experiment, we combined the C1q2 and C2q2 crosslinked samples prior to trypsin digestion and C18 cleaning, and used 1 ug for MS analysis. In both experiments, we identified one monolinked peptide (₃₀₆SYEEELAK*DPR₃₁₆) that was fivefold more abundant in the samples containing maltose compared to the samples without maltose (Figure 2a), suggesting K313 is modified more readily by the Q2linkers when maltose is present. Interestingly, this peptide happens to reside within the ‘balancing interface’ of MBP. In the closed conformation, the sequence between 301 and 312 forms an alpha helix (Figure 2a, gray structure) and upon binding to maltose, the alpha helix relaxes and unwinds (Figure 2a cyan structure). However, K313 itself is solvent exposed in both structures, so the increase in K313 modification in the open conformation cannot be simply explained by an increase in accessibility upon maltose binding. Comparison of the open and closed conformations of MBP reveals that K313 can form a salt bridge with E310 in the open conformation (K313-NE to E310-OE2 distance of ~3.3 Å), and this salt bridge is broken in the closed conformation. It is likely that the salt bridge between K313 and E310 limits the NHS ester-based modification of K313 as revealed by the quantitative difference in the abundance of the Q2linker-modified peptide containing K313.

Calmodulin (CaM), a small polypeptide composed of 148 AAs, is a well-studied biosensor that undergoes conformational changes upon binding to calcium and CaM-binding peptides (CBPs) (Meister and Joshi, 2013). To evaluate the ability of Q2linkers to detect conformational changes in CaM, we crosslinked 25 ug CaM, in the presence or absence of 20 mM CaCl₂ and a nearly 1:1 molar ratio of CBP (8 ug) derived from alphaII-spectrin with C2q2 and C1q2, respectively. After crosslinking, the samples were combined, trypsin digested, and 1 ug of the sample was analyzed by MS. One crosslinked peptide was identified containing a Lys78 to Lys95 linkage, which was fivefold less abundant in the presence of CBP and CaCl₂ (Figure 2b), suggesting that crosslinking between these two residues is greatly inhibited upon calcium and CBP association with CaM. We also identified the corresponding monolinked peptides containing these two residues and found that their abundances were slightly increased upon binding of calcium and CBP (Figure 2b). These results suggest that the reduced abundance of the crosslinked peptide containing the Lys78-Lys95 linkage cannot be explained by limited accessibility of these two sites in the presence of Ca²⁺ and CBP. Indeed, the crystal structures of apo-CaM (1cfd) and CBP-bound-CaM (2bbm) show no changes in the surface exposure of Lys78 and Lys95. However, CBP binds to the region between Lys78 and Lys95 and interferes with the ability of the Q2linker to form a crosslink between them due to either steric hindrance or decreased flexibility of CBP-bound CaM (Figure 2b). The results from the MBP and CaM qCLMS experiments show that the Qlinker approach can detect conformational changes in biosensors.

When proteins interact with one another to form a quaternary protein complex, the complex often will have lower energy states with subtle conformational changes and/or structural rearrangements, albeit many of these changes are unknown and are difficult to detect. We next wanted to see whether Q2linker can be used to detect subtle conformational changes, as well as protein–protein interaction changes during quaternary complex formation. During pol II transcription initiation, general transcription factors, including TBP, TFIIA, and TFIIB, play central roles in promoter recognition, preinitiation complex (PIC) formation, and start site selection (Murakami et al., 2015; Tsai and Sigler, 2000). During PIC formation, TBP binds to TATA-containing promoter DNA, and TFIIA, composed of two subunits in yeast, TOA1 (or TFIIA1) and TOA2 (or TFIIA2) (Ranish and Hahn, 1991), associates with and stabilizes the TBP-TATA DNA complex (Tan et al., 1996; Figure 3c). The C-terminal core domain of TFIIB also interacts with TBP, while the N-terminal zinc ribbon domain and B finger of TFIIB reach into the active center of pol II (Chen and Hahn, 2004). The central linker region of TFIIB (81-211) snakes down the central cleft of pol II, interacting with the Rpb1 clamp and Rpb2 protrusion loops to stabilize the PIC complex (Murakami et al., 2015). TFIIA and TFIIB independently interact with the N- and C-terminal lobes of TBP, respectively; no interaction between TFIIA and TFIIB has been described (Tan et al., 1996; Andel et al., 1999). We incubated purified, recombinant TBP with either TFIIA or TFIIB (1:1 by weight for both reactions) to form TBP-TFIIA and TBP-TFIIB complexes, and then crosslinked each complex separately with C1q2 crosslinker. At the same time, we incubated TBP, TFIIA, and TFIIB (1:1:1 by weight) to form the TBP-TFIIA-TFIIB complex, and crosslinked the sample with C2q2 linker. After quenching the crosslinking reactions, all of the samples were combined, digested with trypsin, fractionated by strong cation exchange (SCX) chromatography, and prepared for MS analysis. SCX was used to reduce the complexity of the peptide mixtures. After database searching with both pLink2 and Nexus algorithms, 61 interlinks were identified. We grouped the interlinks between the different proteins in the complexes and found a tight distribution of log2 (127/126) reporter ion intensity ratios centered around 0 with most ratios between 1.4 to –1.4-fold (Figure 3a). Two crosslinks between TFIIA1 and TFIIB have large abundance differences (>4.0-fold) because crosslinks between these proteins can only be observed in the TBP-TFIIA-TFIIB samples crosslinked with C2q2 (127). TFIIA and TFIIB were not co-incubated in either of the samples crosslinked with the C1q2 (126) crosslinker. 202 intralinks were also identified. Since there is twice as much TBP in the pooled C1q2 crosslinking reactions, the average log2 (127/126) ratio for TBP intralinks is ~–0.5 (~1.4-fold more in the samples crosslinked with the C1q2 crosslinker). We think this is reasonable as the crosslinked peptide ratios are not only related to the protein abundance but also to crosslinking patterns, crosslinking efficiency, monolinks, mis-cleavages, etc. We re-centered the log2 (127/126) ratios for TBP intralinks at –0.5 while leaving the ratios for the other intralinks unchanged to compare the ratio distribution of the intralinks (Figure 3b). Like interlinks, most intralink ratios show no significant changes between the two experimental conditions examined here.

No significant conformational changes have been observed previously based on the crystal structures and biochemical studies of the TBP/TFIIA, TBP/TFIIB, and TBP/TFIIA/TFIIB complexes with TATA DNA (Murakami et al., 2015; Tan et al., 1996; Nikolov et al., 1995; Dion and Coulombe, 2003; Kays and Schepartz, 2000). This is due to the fact that TFIIA and TFIIB independently interact with the N- and C- lobes of TBP, respectively, and no direct interaction between TFIIA and TFIIB is observed in the cryo-EM structures (Murakami et al., 2015; Andel et al., 1999). In addition, only the core, conserved regions of TFIIB, TBP, and TFIIA were mapped in the crystal/cryo-EM structures. We used full-length versions of TFIIB, TBP and TFIIA in our study. Most of the interlinks and intralinks identified in our study have no significant abundance changes between the samples, in agreement with previous structural studies (Figure 3a and b). Interestingly, the largest interlink changes, besides the previously described crosslinks between TFIIA1 and TFIIB, involve crosslinks between the N-terminal region of TBP and the central linker region of TFIIB, both of which are unstructured and absent in the crystal structures. The largest ratio changes among the intralinks all involve the central linker region of TFIIB, whose structure has only been observed in complexes containing pol II (Figure 3c). Our results suggest that the addition of TFIIA to the TBP/TFIIB complex induces a change in the conformation of the TFIIB linker region. This may involve the rearrangement of the TBP N-terminal domain (NTD). Previously, it was suggested that TFIIA stimulates TBP binding to DNA by causing a structural change involving the NTD of TBP, which alleviates an inhibitory function of the NTD (Lee et al., 1992; Bleichenbacher et al., 2003). This TFIIA induced change in the conformation of the TBP NTD may be reflected by the reduced abundance of the intralink between TBP residues 47 and 83 in the sample containing TBP/TFIIA/TFIIB (Figure 3B). Thus, it is possible that the interaction between TFIIA and TBP not only increases TBP’s affinity for TATA DNA, but also causes a structural rearrangement in both TBP and TFIIB so that TFIIB may be better positioned to interact with pol II for PIC formation (Chen and Hahn, 2004; Glossop et al., 2004; Bangur et al., 1999). Our study has captured dynamic structural changes which accompany the formation of the TFIIA/TBP/TFIIB complex.

We next evaluated the ability of Q2linkers to detect conformational changes in large protein complexes. Yeast pol II is a >0.5 MDa protein complex and a good model system to study structural changes in large complexes as the 12-subunit holo-pol II assumes a conformation that is distinct from that of the 10-subunit, core pol II complex, lacking the Rpb4/Rpb7 dimer (Cramer et al., 2001; Bushnell and Kornberg, 2003; Oberthuer et al., 2017). In core pol II (1i3q), the ‘clamp’ is in an ‘open’ state, allowing formation of a straight channel for DNA template entry (Cramer et al., 2001). In the core pol II elongation complex (1nik) and the holo-pol II enzyme (5u5q), a massive movement of the ‘clamp’, which rotates by about 30⁰ with a maximum displacement >30 Å at external sites, results in the ‘closed’ state (Bushnell and Kornberg, 2003; Gnatt et al., 2001). However, most of the clamp moves as a rigid body and the large structural movement is produced by conformational changes in five ‘switch’ regions (Gnatt et al., 2001; Figure 4—figure supplement 1, magenta). The Rpb4/Rpb7 heterodimer binds to core pol II through interactions between Rpb6 (91–105) and Rpb1 (1440–1452) with Rpb7. RPB4 is not required for viability while RPB7 is essential for growth in yeast. We affinity-purified holo-pol II from a yeast strain expressing FLAG-tagged Rpb3, and we purified pol II lacking Rpb4 from an RPB4 deletion strain expressing FLAG-tagged Rpb2 (Figure 4a). We crosslinked ∆Rpb4 pol II and holo-pol II with C1q2 and C2q2, respectively, in experiment I, and then performed a crosslinker swapping experiment in which we crosslinked ∆Rpb4 pol II and holo-pol II with C2q2 and C1q2, respectively, in experiment II (Figure 4a). The samples were analyzed by MS, and Comet database searching against the yeast proteome identified unmodified peptides and monolinks corresponding to 234 proteins (>99% probability). We used pLink2 (Chen et al., 2019) and Nexus (Mashtalir et al., 2018) to identify crosslinks by searching a database composed of the sequences of the 12 pol II subunits, and then extracted the ion intensities for the 126 and 127 reporter ions from the identified spectra for their relative quantification. We then averaged the log2(126/127) ratios from each spectrum corresponding to a Qlinker-modified site or pair of crosslinked sites (Supplementary file 1), and plotted the ratios from experiment I on the x-axis and experiment II on the y-axis of the graphs shown in Figure 4b–d. Each green dot represents one unique site or pair of sites identified in both experiments. Each blue or yellow dot represents a unique site or pair of sites identified only in experiment I or experiment II, respectively.

Here, 101 interlinks were identified in both experiments I and II, 47 interlinks were identified in experiment I only, and 116 interlinks were identified in experiment II only (Figure 4b). However, among all of the interlinks that were only identified in one of the experiments, only three in experiment I and four in experiment II exhibit a greater than two fold intensity difference. This suggests that the inability to identify most of these crosslinked peptides in both experiments is mainly due to undersampling during MS analysis of the complex samples, rather than the absence of the crosslinked peptides in one of the experiments. This also highlights the importance of reliable methods for quantification in CLMS experiments involving complex samples, where undersampling of crosslinked peptides is exacerbated due to the increased complexity of the crosslinker-modified samples and the inefficiency of the crosslinking reaction. Considering all of the interlinks identified in both experiments, there is a rough anti-correlation between the ratios measured in experiment I and II (R² = 0.5) (Figure 4b). We labeled all of the interlinks that exhibit more than a twofold abundance difference in both experiments (Figure 4b). The crosslinks between Rpb4:80-Rpb7:23, Rpb4:76-Rpb7:23 and Rpb1:2-Rpb7:29, labeled in green, were more abundant in the holo-pol II sample as the ∆Rpb4 pol II sample lacks Rpb4 and contains sub-stoichiometric amounts of Rbp7. Several interlinks and intralinks involving Rpb1:332, Rpb2:507, and Rpb1:1102, labeled in red, are also more abundant in holo-pol II (Figure 4b and c). Rpb1:332 is located in the Switch 2 region (Rpb1:328–346), which is the main switch responsible for the conformational change from the ‘open’ to ‘closed’ state (Figure 5a). The helix in the open state (cyan) flips out (gray) toward the cleft to contact DNA at the −2, –1, and +2 positions (Gnatt et al., 2001). This conformational change is accompanied by stabilization of the Rpb2 forkloop 2 (Rpb2:503–508). The conformational change in Switch 2 brings Rpb1:332, Rpb1:1102, and Rpb2:507 closer to each other, allowing them to be crosslinked by Q2linkers (Figure 5a).

The crosslinks between Rpb1:15-Rpb6:76 and Rpb1:15-Rpb6:72, labeled in blue, are influenced by a conformational change in Switch 5 (Rpb1:1431–1433), which undergoes a hinge-link bend (Gnatt et al., 2001; Figure 5b). In the core pol II structure, Rpb1:K15 forms a salt bridge with Rpb1:D1442 (K15:NZ - D1442:OD2 distance of 3.8 Å). In holo-pol II, Switch 5 bending pulls Rpb1:D1442 away from K15, breaking the salt bridge (NZ-OD2 distance of 6.7 Å). Even though the relative positions of Rpb1:K15, Rpb6:K76, and Rpb6:K72 do not change significantly in the holo- and core pol II structures (Figure 5b), the increase in crosslink abundances involving these residues in the ‘open’ state is likely attributed to the salt bridge between K15 and D1442 in the ‘closed’ state, which impedes the NHS ester-based reaction between the epsilon amino group of K15 and the crosslinker. Not all of the crosslink abundance changes can be easily explained. Rpb1:1246 is located in a region that interacts with Rpb9 and Rpb2, and changes from a disordered region (1245–1254) in core pol II to a loop in holo-pol II (Gnatt et al., 2001). The increased abundance of the Rpb1:1246-Rpb9:77 in holo-pol II might reflect this structural change. Rpb10:68 is very close to Rpb2:191, but there is no obvious difference in the location of the residues in both structures. Rpb1:129 is located at the N-terminus of Rpb1 (the major part of the clamp domain 1–346) that moves as a rigid body. A slight orientation difference of Rpb1:K129 may affect its ability to crosslink to Rpb5:K171. The crosslink between Rpb11:37-Rpb1:129 could be a false-positive identification as these residues are located on opposite sides of pol II and the distance between their Cα atoms exceeds the theoretical crosslinking distance of Q2linkers.

Here, 287 intralinks were identified in both experiments, and 124 and 143 intralinks were identified only in experiments I and II, respectively (Figure 4c). The anti-correlation of log2(126/127) ratios for the intralinks identified in both experiments is not strong (R² = 0.3). All of the intralinks involving Rpb4 are more abundant in holo-pol II sample (even though we do not expect any reporter ion signal from Rpb4 peptides derived from the ∆Rpb4 pol II sample, we still observed reporter ion signals from the channel corresponding to the ΔRpb4 sample, potentially due to the presence of low abundance, co-eluting ions). In this study, the intralinks are less useful for probing conformational changes as the backbones of most subunits align pretty well in both complexes, and there are only small rearrangements for most residues. For example, the Rpb5:161–171 crosslink is enriched in the ∆Rpb4-pol II sample, but there is little structural change for the region involved (Figure 5c). Rpb5:K161 overlays perfectly in both structures and forms a salt bridge with Rpb5:E172 with NZ-OE2 distances of 2.7 Å and 2.8 Å in the holo- and core pol II structures, respectively (Figure 5c). Rpb5:K171 assumes slightly different orientations in both structures due to the formation of a helical structure between residues 171–175 in holo-pol II (Figure 5c). Rpb5:K171 is slightly more stable in core pol II (cyan, b-factor 74) than in holo-pol II (gray, b-factor 216), which may account for its increased abundance in the ΔRpb4-pol II sample. The Rpb1:689–728 crosslink is enriched in the holo-pol II sample, while these two sites, located in the middle of two alpha-helices, are well-aligned (Figure 5d). The distance between the two NZ atoms of the lysine residues is ~4 Å in core-pol II and ~6.6 Å in holo-pol II. The 4 Å distance in the ΔRpb4-pol II sample may limit the ability of the Q2linkers to react with both ε amines to form a crosslink between the two lysine side chains. Also, 191 monolinked peptides were identified in both experiments and 10 and 11 monolinked peptides were only identified in experiment I or II, respectively. All of the Rpb4 and Rpb7 monolinks are enriched in the holo-pol II sample as expected. Some Rpb1 residues at the N-terminal clamp domain were also enriched in the holo-pol II sample (Figure 4d). In general, and like intralinks, the monolinks are less informative in this study for probing conformational changes. Interestingly, some Rpb5 monolinks and intralinks are slightly enriched in the ∆Rpb4-pol II sample, even though there is no large conformational or structural rearrangement associated with this region. Rpb5 interacts with the same domains of Rpb6 and Rpb1 that interact with Rpb4 and Rpb7. It is possible that Rpb4/7 stabilizes Rpb1, Rpb6, and Rpb5 and their interactions with one another so that the region is more rigid in holo-pol II and less reactive to Q2linkers.

Di-tert-butyl 3,3'-iminodipropionate was purchased from TCI America (Portland, OR). Bromoacetic acid 1-¹³C and 2-¹³C were purchased from Cambridge Isotope Laboratories, Inc (Tewksbury, MA). The N,N’-diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIPEA), dimethylformamide (DMF), acetonitrile (ACN), dichloromethane (DCM) 2,6-dimethylpiperidine, N,N,N’,N’-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), Human Brain Calmodulin, and bovine serum albumin (BSA) were purchased from MilliporeSigma (St. Louis, MO). Calmodulin binding peptide 1 (alphaII-spectrin peptide) was purchased from GenScript Biotech Corp. (Piscataway, NJ). Bacterial maltose binding protein was purchased from Novus Biologicals (Littleton, CO). NGC chromatography system from Bio-Rad Laboratories (Hercules, CA) was used for reverse-phase FPLC-C18 separation, and SNAP ULTRA C18 flash cartridges (40 g) were purchased from Biotage (Uppsala, Sweden).

To synthesize the Qlinker, we first synthesized (2,6-dimethylpiperidin-1-yl) acetic acid and reacted it with di-tert-butyl-3,3’-iminodipropoinate (‘1’) but we obtained little desired product due to steric hindrance of dimethylpiperidine, resulting in difficulties activating the (2,6-dimethylpiperidin-1-yl) acetic acid. Then we used a peptoid synthesis strategy to have a one-pot synthesis of di-tert-butyl protected product ‘2’ using the 1-¹³C or 2-¹³C bromoacetic acid (BAA) for the two isobaric crosslinkers (Figure 1—figure supplement 1). 0.5 mL di-tert-butyl 3,3'-iminodipropionate (‘1’) (~3.2 M, 1 eq), 0.5 ml DIC (~6.4 M, 2 eq), and 280 mg bromoacetic acid 1-¹³C or 2-¹³C (1.25 eq) in 1 ml DMF were mixed for 2 hr at room temperature (RT). Then 10 ml DCM and 10 ml 0.1% trifluoroacetic acid (TFA) were used to extract product di-tert-butyl-3,3'-[(bromoacetyl)azanediyl]dipropanoic acid in the DCM phase. 1.1 ml (~5 eq) dimethylpiperidine was added to the DCM extract and mixed at RT for 2 hr, and the solvent was then evaporated under vacuum. The product di-tert-butyl-3,3'-{[(2,6-dimethylpiperidin-1-yl)acetyl]azanediyl}dipropanoic acid ‘2’ was then dissolved in 50% ACN/0.1% TFA and diluted to 20% ACN. The precipitates were removed by centrifugation and the supernatant was loaded onto a FPLC-RP-C18 cartridge at 5 ml/min and eluted with a 70 min gradient from 20 to 60% ACN. The product was monitored by 215 nm absorption and confirmed by MS analysis using an LTQ (Thermo Scientific). The fractions containing the product ‘2’ were then combined and evaporated by rotovap. 5 ml TFA was then added to the dried substance for 2 hr and then evaporated by rotovap. The final product ‘3’ of 3,3'-{[(2,6-dimethylpiperidin-1-yl)acetyl]azanediyl}dipropanoic acid was then dissolved in 0.1% TFA, loaded onto an FPLC-RP-C18 column, and eluted with a 50 min gradient from 0 to 20% ACN. Product ‘3’ with MH+ of 316.34 was collected and was determined to be >95% pure. Product ‘3’ was fractionated a second time using the same FPLC-RP-C18 conditions to yield ~50 mg of the final product at ~99% purity (~10% efficiency). In situ activation of Q2linker was achieved by mixing 0.25 M product ‘3’ dissolved in dry DMF with an equal volume of 0.5 M TSTU in dry DMF and 1/10th volume of DIPEA at RT for 1 hr. The final stock concentration of Q2linkers was ~0.12 M, and the crosslinkers can be stored at –20°C or –80°C for up to 1 month with little loss of activity. Hydrolysis of the activated crosslinker was observed upon longer storage, possibly due to trace amounts of water in the DMF solution.

To perform the crosslinking reaction, we typically used freshly activated crosslinkers or brought the frozen crosslinkers to RT for 30 min before use. A typical crosslinking reaction is carried out in 50 mM HEPES buffer (pH 7.9) or 1× PBS (pH 7.5) with 20–100 μg total protein in a volume of 50–400 μl with ~2 mM (50× dilution from the stock) crosslinker. Nearly equal amounts of proteins were first crosslinked with one of the Q2linkers for 1–2 hr at RT and then 10 μl 1 M ammonium sulfate was added to quench each reaction for 10 min. The two crosslinked samples were then combined and vortexed to mix the sample. 10 μl 20 mg/ml SP3 magnetic beads were added to the sample and the sample was mixed well. ACN was then added to 70%, and the sample was incubated in a thermomixer at 60°C for 30 min with mixing. After collecting the beads on a magnetic stand, the beads were washed with 100% ACN and resuspended in 100 μl 8 M urea, 50 mM choloroacetamide (CAA), 50 mM TCEP in 1 M ammonium bicarbonate buffer at 37°C for 1 hr. The beads were then diluted by adding 700 μl pure water and 1/10 (w/w) trypsin to digest the sample overnight at 37°C with rotation. The digested peptides were then purified using C18 micro-tips and dried for MS analysis. Complex samples were fractionated by HPLC using in-house prepared microcapillary SCX columns (200 μm × 20 cm; SCX 3 μm, Sepax Technologies) at a flow rate of 2–3 μl/min. Peptides were eluted with 20 μl of Buffer A (10% ACN, 0.1% FA) containing 30, 50, 70, and 100% Buffer B (800 mM ammonium formate, 20% ACN, pH 2.8), followed by 50 μl Buffer D (0.5 M ammonium acetate, 30% ACN). All fractions were dried in a Speed-vac and resuspended in 0.1% TFA and 2% ACN.

We used the C1q2 and C2q2 reagents to crosslink BSA and analyzed the sample using a Thermo Oribtrap-Fusion mass spectrometer with an HCD collision energy of 28 or 30%. The monolinked peptide spectra were readily identified by Comet database searching using a differential modification of 297.1770 on lysine residues, but most spectra had very low or no reporter ion intensities. We then increased the HCD collision energy to 35, 40, and 45% and observed higher reporter ion intensities, with 35% energy giving both reasonable fragmentation patterns and high reporter ion intensities, and collision energies above 35% resulting in poor fragmentation spectra and significantly reduced the numbers of identified peptides (data not shown). Synchronous precursor selection coupled with MS3 (SPS-MS3) technology can be used to quantify TMT-labeled peptides because all of the b ions and lysine containing y ions are TMT-labeled and can be selected for MS3 to generate reporter ions at high energy (McAlister et al., 2014). However, this technology cannot be used for Q2linker quantification because most fragment ions will not retain the reporter moiety and thus will not yield reporter ions during MS3. At the same time, the Q2linkers are only associated with modified lysine residues and may not produce observable daughter ions in the MS2 spectra that can be selected for MS3, especially for the crosslinked species. The Yates group reported that a stepped HCD strategy increased both the diversity of fragmentation ions and TMT reporter ion intensity (Diedrich et al., 2013). Following their suggestion for TMT-labeled peptides, we used stepped HCD settings of 24, 30, and 36% and obtained MS2 spectra with much higher reporter ion intensities as well as better fragmentation across the peptide backbone. The average reporter ion intensity under this condition is about 60% ± 25% of the highest intensity peaks. Thus, this condition was used for all subsequent MS analyses of Q2linker-crosslinked samples.

Expression plasmids containing His6-tagged versions of TBP, TFIIA, and TFIIB were transformed into BL21 (DE3)-CodonPlus-RIL cells (Agilent, Santa Clara, CA) in LB broth with carbenicillin/kanamycin and chloramphenicol. Protein expression was induced by addition of IPTG to 0.15 mM for TFIIA/TFIIB and 0.5 mM for TBP at 37°C for 4 hr after OD600 reached 0.3–0.5. Cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.5, 250 mM KCl, 10% glycerol, 10 mM βME, 10 mg/ml lysozyme) for 30 min at 4°C and then sonicated using a Superhorn sonicator with output 9 in an ice-water bath using 1 min cycles of 30 son, 30 s off, over 10 min. The lysates were clarified by centrifugation at 30,000 × g for 20 min and proteins were purified using HisPur Ni-NTA resin (Thermo Fisher Scientific). The proteins were eluted with 200 mM imidazole and concentrated using 3K MWCO Amicon Ultra 4 ml spin columns followed by buffer-exchange to reaction buffer (20 mM HEPES-NaOH, pH 7.9, 100 mM KOAc). 25U of SUMO protease (Invitrogen) was used to cleave the N-terminal His-sumo tag on TFIIB at 4°C overnight and HisPur Ni-NTA was used to remove the SUMO protease and the His6-sumo tag.

20 ug of each purified protein was incubated in 100 ul reaction buffer for 30 min at RT in the following combinations: TBP/TFIIA, TBP/TFIIB, and TBP/TFIIA/TFIIB. Then C1q2 crosslinkers were added to reactions containing TBP/TFIIA and TBP/TFIIB and C2q2 crosslinkers were added to reactions containing TBP/TFIIA/TFIIB at ~1 mM for 2 hr before quenching with 5 ul 1 M ammonium bicarbonate. The samples were combined and 1/10th volume of 10% sodium deoxycholate (SDC) was added followed by addition of TCEP and CAA to 10 and 25 mM, respectively. The proteins were denatured at 95°C for 10 min and then diluted to 0.5% SDC for trypsin digestion overnight. The SDC was precipitated by addition of 1% TFA and centrifugation at 16,000 × g for 10 min. The peptides were C18 cleaned and fractionated by HPLC-SCX prior to MS analysis.

We constructed yeast (Saccharomyces cerevisiae) strains carrying C-terminal His6-3XFLAG-His6-Ura3 (HFH) tags on RPA2 and RPB3 by swapping the tandem affinity purification (TAP) tag for the HFH tag in strains carrying C-terminal TAP tags on RPA2 and RPB3. The RPB2-HFH strain was generated by transforming a ∆rpb4 strain with a PCR product containing the HFH tag with 40 bps of sequence flanking the stop codon of RPB2 gene. For each complex purification, we normally grew 6 l of the appropriate yeast strains in YPD media overnight to OD600 11–13. The cells were harvested by centrifugation and frozen in liquid N₂. After evaporation of the liquid N₂, the cell pellets were ground to a fine powder in a coffee grinder. 40 ml lysis buffer (50 mM HEPES, pH 7.9, 400 mM ammonium sulfate, 10 mM MgSO₄, 1 mM EDTA, 20% glycerol) with protease inhibitors was then added to the fine powder and the mixture was stirred at 4°C for 1 hr. The cell lysate was then sonicated using a Superhorn sonicator with output 9 in an ice-water bath using 1 min cycles of 30 s on, 30 s off, over 10 min. The lysate was centrifuged at 20,000 × g for 1 hr and the supernatant was then mixed with 2 ml anti-FLAG M2 affinity agarose (Sigma-Aldrich) overnight at 4°C with rotation. The beads were then collected in a column and washed with 40 ml lysis buffer twice, 40 ml 2× PBS buffer, and 40 ml 1× PBS with 0.1% NP-40. The complexes were eluted with 3X FLAG peptide (Sigma-Aldrich) at 0.4 mg/ml in 1× PBS. The complexes were then concentrated by repeated centrifugation and dilution with 1× PBS (normally three times) in Amicon Ultra-4 devices (100K cutoff, Millipore) to reduce the concentration of the 3X FLAG peptide. After a final centrifugation step, protein concentration was determined by Qubit protein assay (Thermo Fisher Scientific). We usually isolated ~200–300 μg protein from a 6 l culture. Higher yields of pol II were obtained from the ∆RPB4 strain. We checked the purity of the sample and subunit composition by Coomassie stained SDS-PAGE.

Peptides were analyzed by electrospray ionization microcapillary reverse-phase HPLC with a column (75 μm × 270 mm) packed with ReproSil-Pur C18AQ (3 μm 120 Å resin; Dr. Maisch, Baden-Würtemburg, Germany) on a Thermo Scientific Fusion with HCD fragmentation and serial MS events that included one FTMS1 event at 30,000 resolution followed by FTMS2 events at 15,000 resolution. Other instrument settings included MS1 scan range (m/z): 400–1500; cycle time 3 s; charge states 3–8; filters MIPS on, relax restriction = true; dynamic exclusion enabled: repeat count 1, exclusion duration 30 s; filter IntensityThreshold, signal intensity 50,000; isolation mode, quadrupole; isolation window 3 Da; activation type: HCD; collision energy mode: stepped; HCD collision energy (%): 24, 30, 36; AGC target 500,000, max injection time 200 ms. HPLC uses an 80 min gradient from 10% ACN to 40% ACN.

The RAW files were converted to mzXML files by Rawconverter (He et al., 2015). For normal peptide and monolinked peptide searches, we used the Trans-Proteomics Pipeline (TPP)/Comet searches (http://tools.proteomecenter.org/wiki/index.php?title=Software:TPP) with static modification on cysteines (+57.0215 Da) and differential modifications on methionines (+15.9949 Da) and lysines (+297.166962 Da and +279.166397 Da). Spectra identified as peptides with lysine modification(s) with PeptideProphet probability >95% were output as spectra for monolinked peptides. For crosslinked peptide searches, we used two crosslink database searching algorithms: pLink2 (Chen et al., 2019) and an in-house-designed Nexus (Mashtalir et al., 2018) with Q2linker mass of 279.1664 Da against a database containing only yeast pol I or pol II protein sequences and its reverse decoy database(s). Other searching parameters include precursor monoisotopic mass tolerance: ±20 ppm; fragment mass tolerance: ±20 ppm; up to three miscleavages; static modification on cysteines (+57.0215 Da); differential oxidation modification on methionines (+15. 9949 Da), peptide N-terminal glutamic acid (–18.0106 Da), or N-terminal glutamine (–17.0265 Da); and Q2linker modification on lysines (+297.166962 Da). After performing the pLink2 and the Nexus analyses with 5% FDR, the search results were combined and each spectrum was manually evaluated for the quality of the match to each peptide using the COMET/Lorikeet Spectrum Viewer (TPP). The crosslinked peptides are considered confidently identified if at least four consecutive b or y ions for each peptide are observed and the majority of the observed ions are accounted for. Search results that did not meet these criteria were removed. The spectra that passed our evaluation are summarized in Table S1 and are uploaded into ProXL (Riffle et al., 2016) for viewing and data analysis. All of the data, including the spectra, linkages, and structural analyses, can be visualized at https://www.yeastrc.org/proxl_public/viewProject.do?project_id=634. The raw files are deposited at protomeXchange: PXD035939 and PXD056825.

A Perl script is used to extract the 126 and 127 reporter ion intensities for the identified monolinked- or crosslinked-spectra from mzXML files within a mass tolerance of 0.005 Da (~40 ppm) of the theoretical 1+ mass of 126.127726 and 127.131081 for the 126 and 127 reporter ions, respectively. These observed intensities are designated I126 and I127, respectively. Like TMT quantification, these observed intensities need to be adjusted based on the natural distribution of monoisotopic elements and the 99% purity of the heavy 13C element. It is relatively simple for the Q2linkers since only two channels need to be considered. We corrected the reporter ion intensities (A126 and A127) by solving the equations: A126 * 0.90754+A127 * 0.0 1 = I126 and A126 * 0.09246 + A127 * 0.90724 = I127. The final intensities A126 = (I126 - I127*0.01102)/0.906521 and A127 = (I127 - I126 * 0.10188)/0.906221 were used to calculate the log2 ratios of the126 and 127 reporter ions. If multiple spectra were identified for a crosslinker-modified site, we used the average log2 ratio of all the identified spectra corresponding to that site.

Average and boxplot in R were used to generate Figure 1d and e. Crosslinker swapping experiments were performed for the experiments presented in Figures 2a and 4.