2.1 Definition of autoinhibited and active states from X-ray crystallography

The SFKs possess a conserved motif of structured domains that permit control of their Tyr kinase activity. Structural elucidation with X-ray crystallography of c-Src [33,34] and HCK [35,36] generated the first snapshots of the autoinhibited state. A consensus activation mechanism based on these structures indicates that SFK activation proceeds via unlatching of the autoinhibited complex clamped into this conformation by phosphorylation at Tyr-527 on the C-terminal tail[9,37], which is regulated by activity of the C-terminal Src kinase (CSK) (Fig. 1A and B)[38]. The intramolecular domain-domain interactions maintain the kinase domain in the inactive configuration. Release of these interactions is accomplished through a combination of events that can involve SH3 competitive binding of poly-proline motifs in peptides[39], dephosphorylation of pTyr-527, and/or competition with a tighter binding pTyr-containing peptide fragment (Fig. 1C). “Unclamping” of the SH2 and SH3 domains alleviate the autoinhibiting constraints from the “back” of the kinase domain allowing for structural flexibility to convert to the active state (Fig. 1D)[37,40]. The most important structural changes involve unfolding of the kinase A-loop (orange), which exposes Tyr-416 for autophosphorylation [34] and the resulting phosphorylated tyrosine facilitates permanent A-loop unfolding, accessibility of substrates to the ATP site for phosphoryl transfer, and inward rotation of the ɑC-helix (red)[41]. In more detail, the folded A-loop (orange in Fig. 1E) holds back the ɑC-helix in the inactive conformation and Glu-310 is positioned toward solvent. Upon A-loop unfolding, promoted by breaking the SH2 and SH3 domain-domain interactions, the ɑC-helix has the open space to rotate inward establishing the activating salt bridge between Lys-295 and Glu-310 (Fig. 1F). These insights from crystal structures of Src and other SFKs indicate that their activation is complex involving large-scale conformational changes mediated through intramolecular domain-domain interactions and changes in phosphorylation of pertinent tyrosine residues[9]. Understanding the nature of these 3-dimensional SFK structures are critical when discussing the potential roles of reactive cysteine residues and rationalizing their susceptibility to oxidation.

Fig. 1

Structural basis for activation of the non-receptor Src family kinases from X-ray crystallography. A) The sequence of all SFKs contain a “Src module” consisting of consecutive encoding of structured SH3, SH2, and a kinase domains following a unique sequence in each SFK. Generally, two regulatory tyrosines (Y416 and Y527 in the chicken Src sequence numbers) serve as targets of phosphorylation & dephosphorylation. B) The Autoinhibited (closed) structure of Src (PDB code 2SRC) contains the Src module folded into a compact structure with SH2 an SH3 domains maintaining the kinase domain in the inactive conformation dependent on dephosphorylation at Y416 and phosphorylation at 527. C) Activation of Src is multifaceted and can proceed via any combination of engagement of SH2 with phosphopeptides, competition of the SH2-kinase linker with exogenous polyproline peptide motifs (-XPpXP-), and dephosphorylation at pY527. D) Structure of the active kinase domain containing unfolded, phosphorylated A-loop (pY416) and inward rotated αC-helix (PDB ID 3DQW). E) View of catalytic cleft of autoinhibited versus (PDB ID 2SRC)) and F) active Src (PDB ID 3DQW) showing the structural consequences of A-loop unfolding and the formation of the Lys-295 to Glu-310 H-bond via inward rotation of the αC-helix.

2.2 Characterizing the intermediates of SFK activation from computational modeling

As X-ray crystal structures represent distinct states, more detailed understanding has been afforded by computational modeling of Src with molecular dynamics (MD) simulations specifically motivated to elucidate the intermediate structures along the trajectory of activation and expose their vulnerability to inhibition for drug discovery[[41], [42], [43], [44]]. Results from such MD simulations indicate that the isolated Src kinase domain undergoes transition from inactive to active states through two key intermediates that involve first transitional unfolding of the A-loop (I₁) followed by partial rotation inward ɑC-helix (I₂)[42]. These findings indicate that the late-stage ɑC-helix inward rotation is ultimately most consequential for SFK activity as compared to the A-loop unfolding[42]. Further work indicates that the A-loop is considerably flexible while Y416 is unphosphorylated where the kinase can sample the active state, however only transiently[44]. Upon phosphorylation, pY416 alters the stability of the kinase and locks it into the active state that is catalytically active and consistent with structures presented previously (Fig. 1)[44]. Overall, these findings are consistent with a stepwise conversion of Src from the inactive autoinhibited state first into an “active-like” state where the A-loop is unfolded but not yet phosphorylated. While unfolded, the A-loop is exposed and can be transphosphorylated to arrive at the fully active state allowing for inward ɑC-helix rotation (see Fig. 1E versus 1F). Subsequent experimental studies have reinforced this notion that several intermediates are accessed in the activation of the non-receptor tyrosine kinases through large-scale dynamic allosteric networks[45,46].

2.3 Biological insights into SFK activation through cell-based studies

The molecular-level details described above from X-ray crystallography and MD simulations are especially valuable when considering measurements from cell-based studies. SFK activation can be interrogated in cells with a number of techniques, such as blotting for phosphorylation status, but considerable insight into activation and resulting structural changes in cells has benefited from the development of FRET-based biosensors (e.g., Src [47], Fyn [48,49], Lck [[50], [51], [52]]), which allow for interrogation of SFKs in “opened” versus “closed” conformations and changes upon stimulation. Importantly, FRET-based biosensors are useful as they revealed properties otherwise hidden from biochemical settings, such as how certain Src inhibitors impact localization as well as stabilize "open" versus "closed" conformation in cellular settings [47]. While monitoring “closed” conformations of SFK appear to reflect autoinhibited or “latched/clamped” SFKs (Y416/pY517), the “open” conformation can represent a series of states depending on the intermediates accessible, as explained from MD simulations, in addition to the fully active pY416 "open" form. A key case that has been especially revealing is for the activation of Lck through T-cell receptor signaling[53]. Interestingly, resting T-cells have been found to contain 50% of Lck in an intermediate “primed” conformation where Tyr-416 and Tyr-527 (Tyr-394 and Tyr-505 in Lck sequence numbers) are not phosphorylated[54]. This “primed” state of Lck, however, appears to not be the active form as recent studies on Lck in T-cell activation demonstrated that phosphorylation at Lck pY416 is required to elicit full T-cell signaling[51,52]. This indicates that the “primed” form of Lck is presumably indistinguishable from the fully extended “open” conformation in FRET-based biosensor measurements[51], supporting the notion that “primed” Lck may be one of the intermediate states seen in MD simulations (I₁/I₂) that is not fully active with a partially unfolded A-loop[[41], [42], [43]]. Utilization of FRET-based SFK biosensors, to my knowledge, have yet to be applied to SFK redox-regulation and may offer key insights into the structural dynamics and cysteine oxidation in response to cellular stimuli.

In contrast, the situation pertaining to Fgr activation in cells appears uniquely atypical involving non-canonical activation independent of SH2 and SH3 “unclamping” potentially due to sequence differences in the SFK A-loops[55]. A non-conserved NPC-motif in the Fgr A-loop (the three amino acids immediately following Y416 where in all other SFKs these amino acids are conserved in a TAR-motif) is thought to be responsible for this idiosyncratic behavior, although further studies appear necessary to confirm this mechanism more fully[55]. These findings demonstrate the complicated nature of SFK activation in their particular cellular settings further motivating efforts to compare and contrast their activation mechanisms and the differences associateed with their redox-active cysteine residues.

3.1 Cys-185

In the autoinhibited complex, the Src C-terminal tail binds to the central β-sheet, positioning the pTyr-527 moiety within a pocket near Cys-185 and other hydrophilic amino acid sidechains (Fig. 2B). Entirely unique to Src, Cys-185 is located on the βC-sheet in a position otherwise conserved as a serine, the hydrophilic alcohol analogue of Cys (Fig. 3)[57]. Of the residues within this region, the conserved cationic Arg-175, and to a lesser extent Arg-155, is principally responsible for binding pTyr-527 and also within H-bond distance of Cys-185[57,58]. Loss of pTyr-527 binding opens this region of the SH2 domain rendering Cys-185 accessible to solvent upon Src activation[59].

Based on the local structural environment around Cys-185, it is straightforward to rationalize how this residue is redox-active due to solvent accessibility and presence in a hydrophilic region including the cationic Arg-175 (Fig. 2B). Indeed, accumulated evidence from a variety of studies have shown that Cys-185 likely exhibits a low pK_a near physiological pH[60], is shown to be involved in disulfide bonding with cortactin at Cys-112/246[61], and most recently readily oxidized by H₂O₂ to sulfenic acid that is involved in the activation of the Src kinase activity[21]. Correspondingly, Cys-185 was found to be highly oxidized (>20%) in the Oximouse database of which was observed predominantly in the subcutaneous fat and elevated with age (Fig. 3C)[26]. Notably, of all SFKs cysteine residues analyzed in oximouse, Cys-185 represents the most highly oxidized cysteine (>50% in old mice subQ).

3.2 Cys-222

Another rare Cys found in a single SFK is Cys-203 in LYN (222 in avian Src, Fig. 2C). This solvent exposed residue located on a short loop connecting βF and ɑB-helix on the opposite of the domain from the pTyr binding site (Fig. 2C). This cysteine is found oxidized in Oximouse generally more so in the younger mouse. While LYN is recognized to be redox-active [62,63], this specific residue has not been addressed previously. It is noteworthy that both Cys-185 and Cys-222 are unique in their respective SFK in positions that mostly contain hydrophilic Ser or Thr residues.

3.3 Cys-238 and Cys-245

The two completely conserved cysteine residues in the SH2 domain are located near the C-terminus (Fig. 2D and E), and a sequence alignment of reveals a conserved (D/A)GLCXXLXXXCX motif[64]. Structurally, both of these residues are on flexible loops that bookend the βG-sheet, and are generally accessible by solvent. Crystal structure coordinates indicate that both residue side chains point away from solvent, and the major difference between these residues is the local proximity of hydrophilic versus hydrophobic residues in the case of Cys-238 and Cys-245, respectively. Oxidation of these residues is observed in Oximouse, but only in a subset of SFKs (Fig. 3). Relative percent oxidation indicates that Src is more oxidized at Cys-238 both in the brain and lung, but is considerably higher in older mice compared to young, while on the other hand, Src Cys-245 is oxidized within the brain and lung, but at lower levels compared to YES1 and FYN in young and old mice, respectively.

4.1 Cys-277 (P-loop)

The N-lobe of the kinase (260–338) contains a single cysteine located on the P-loop (or G-rich loop) that connects β1 and β2 (Fig. 4B). This position is a Cys in a minority of SFKs while otherwise conserved as a Gln (Fig. 5B). Fully exposed to solvent, Cys-277 has been the target of various covalent inhibitors of Src, YES, FGR[71,73], in addition to being the focus of the redox-regulation of Src[21,74,75]. In the later context, oxidation of Cys-277 to a sulfenic acid is uniquely positioned opposite the folded A-loop in the autoinhibited structure, and oxidation to a sulfenic acid promotes the opening of this loop in the redox-regulation of Src[21]. The Oximouse database indicates some levels of oxidation at Cys-277 in both Src and YES1, which is enhanced with age.

Fig. 4

Structure of the Src Kinase Domain and CysteineStructuralEnvironments. A) Overall fold of the Src kinase domain (light blue), ɑC-helix (red), A-loop (orange) in complex with AMP-PNP defining the ATP-binding site from PDB ID 2SRC. Local structural environment for B) Cys-277, Cys-342, and Cys-345 near the ATP binding site and folded A-loop. C) Local hydrophobic residues around Cys-400. D) Placement of Cys-438 near hydrophilic residues, Tyr-416, and Glu-415 of the A-loop in the active kinase (PDB ID 1Y57). E) and F) Organization of the Cys-483, Cys-487, Cys-496, and Cys-498 cluster located near the C-terminal ɑH-helix. In cases where Cys residues are not present in the 2SRC kinase domain, the corresponding position has been mutated to a cysteine in PyMol. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Sequence homology of cysteine residues of SFK kinase domains and percent oxidation. A) Secondary structural elements of SFK kinase domains and location of cysteine residues (colors consistent with Fig. 4). B) Sequence homology of residues at positions of cysteine residues. Two residues are specified if there is a difference between human and mouse sequences. The right and left most residues corresponds to human and mouse, respectively. C) Visualization of percent oxidation from Oximouse. Abbreviations (BAT, brown adipose tissue; epi, epididymal fat; subQ, subcutaneous fat; skm, skeletal muscle; N.D., not detected). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4.2 Cys-342 & Cys-345 (Hinge region & front pocket)

Two non-conserved cysteine residues reside on the hinge region. Cys-342 of FGR is nearest the ATP binding site and the side chain points away from the catalytic binding site into solvent (Fig. 4B). Redox activity of FGR cysteines has yet to be explored, to my knowledge, but is found oxidized in the Oximouse. This position in the other SFKs is a mixture of hydrophilic and hydrophobic amino acids. On the other hand, Cys-345 of BLK is also located on the kinase hinge region near the ATP binding site, otherwise known as the front pocket position. This position of BLK, otherwise a conserved Ser, has also been unexplored previously and is structurally-related to the position of Cys-797 of EGFR [65,66], which is the target of clinically approved TKIs in cancer[76]. Oximouse shows low levels of oxidation of this residue are found in the mouse spleen.

4.3 Cys-400

Located on the β8-helix, directly below the catalytic cleft, Cys-400 is a completely conserved cysteine residue within the SFKs (Fig. 4C), and according to the Oximouse database, this residue is observed oxidized in nearly all SFKs (Fig. 5). Somewhat puzzlingly, the local structure around Cys-400 does not exhibit the typical structural elements of a reactive cysteine residue. Specifically, the region of the kinase domain where Cys-400 is located is structurally ridged, comparing active and inactive structures, in addition to being mostly obscured away from solvent and surrounded by hydrophobic residues. The potential impacts of oxidation on kinase function are also unclear as this location within the kinase domain is far from regulatory segments or domain-domain interactions[45,46]. Biochemical studies are necessary to more completely understand the apparent redox sensitivity of this highly conserved cysteine residue within the SFKs, especially motivated by its universal oxidation in vivo.

4.4 Cys-438

Unique to LYN, Cys-438 is located on a solvent exposed loop in between the ɑEF-helix and ɑF-helix within the vicinity of hydrophilic residues (Fig. 4D). Strikingly, this residue is found highly oxidized in the liver, and previously uncharacterized reactive LYN cysteine[62,63]. The location of this redox-active cysteine is especially intriguing due to its proximity to the unfolded A-loop of the active state[59]. Although potential interactions with anionic Glu-415 may dampen some reactivity of Cys-438, the impact of this unique cysteine on LYN activity may very well be important for kinase activation.

4.5 Cys-483, Cys-487, Cys-496, and Cys-498

A cluster of cysteine residues occurs near the C-terminus of the C-lobe that have garnered considerable attention for their potential roles in SFK redox regulation[16,17]. Cys-498 is conserved in all SFKs and present in >95% of protein Tyr kinases within a MXXCW motif[77]. Cys-487 is also highly conserved present in an extended CXXXXXXXMXXCW motif in 80% of protein Tyr kinases[77]. Two cysteines are also found in this cluster, Cys-483 and Cys-496, which are less conserved. Structurally, this cluster of residues decorates the base of the C-lobe around the ɑH-helix (Fig. 4E and F). Cys-483 and Cys-487 on the loop preceding the ɑH-helix are generally solvent exposed. Cys-496 on the ɑH-helix, and unique to Src, is on a turn of the helix that points the side chain into solvent, whereas Cys-498 is positioned within the four-helix bundle completely obscured from solvent. These cysteine residues occur in a structured region and the conservation of these residues has lead to proposals for their involvement in structural and functional roles across protein Tyr kinases[15,[78], [79], [80]]. However, despite evidence for their oxidation, the impact of these cysteines on SFK function remains unclear (Table 3). One indication may come from a recent study of Src solution dynamics that suggests the conserved Gly-449 on the ɑE-Helix directly across from Cys-498 is necessary for Src activation implying that structural changes to these cysteine residues may impact kinase domain dynamics[46]. Indeed, studies of SFK activation by HgCl₂ and S-nitrosylation appear to react with cysteines in this motif[63,81]. Despite this degree attention paid to these cysteines in the SFKs with respect to redox regulation, very little oxidation is observed in this cluster in the Oximouse database. Oximouse detects significant degrees of oxidation at Cys-487 in LYN and HCK while other forms of oxidation in other SFKs at these residues are not detected. This apparent disparity between Oximouse and the literature highlight current needs with respect to sorting out the importance of the cysteine residues within this C-terminal region of the SFK kinase domain.

Table 3

Modifications and functional outcomes of SFK cysteine oxidation and detected oxidation in young and old mice from oximouse.

	Cysteine Residue1	Modification (Functional Outcome)	Tissue Location of Oxidized Cysteine in Oximouse [26]2
			Young Mice	Old Mice
Src	Cys-185	Disulfide (Cortactin Binding) [61]	Brain, Epi, Kidney, subQ	Brain, Epi, Lung, subQ
		Sulfenic Acid (Activating) [21]
	Cys-238	NR	Brain, Lung	Brain, Lung
	Cys-245	Proposed Disulfide (Activating) [92]	Brain, Lung, Spleen	Brain, Lung
		ND (Activating) [75]
	Cys-277	Sulfenic Acid (Activating) [21]	Lung	Kidney, Lung
		Disulfide (Inactivating) [74]
	Cys-400	NR	Brain, Epi, Kidney, Lung	Brain, Epi, Kindey, Lung, Spleen
	Cys-483	ND (Activating) [93]	Not detected	Not detected
	Cys-487	ND (Activating) [75,93,94]	Not detected	Not detected
		Disulfide (Activating) [92]
	Cys-496	ND (Activating) [93]	Not detected	Not detected
	Cys-498	ND (Activating) [75,93] S-nitrosylation (Activating) [81]	Not detected	Not detected
	Non-identified	ND (Activating) [95,96]
		Sulfenic Acid (Activating) [87,97]
		S-glutathionylation (Activating) [87]
YES1	Cys-238	NR	Brain	Not detected
	Cys-245	NR	BAT, Epi, Heart, Kidney, Lung	BAT, Brain, Epi, Lung
	Cys-277	NR	Kidney, Liver	Kidney, Liver, Lung, Spleen
	Cys-400	NR	Brain, Liver, Lung, skm	Brain, Epi, Heart, Lung, Spleen
FYN	Cys-245	NR	Brain, Spleen	Brain, Heart, Kidney, Liver, Spleen
	Cys-400	NR	Epi, Lung	Brain, Epi, Lung
	Cys-487	ND (Activating) [98]	Not detected	Not detected
	Not specified	Sulfenic Acid (Activating) [99]
FGR	Cys-342	NR	Spleen	Spleen
BLK	Cys-345	NR	Spleen	Spleen
LYN	Cys-222	NR	Brain, Kidney, Lung, Spleen	BAT, Epi, Kidney, Liver, Spleen
	Cys-400	NR	BAT, Brain, Kidney, Lung, skm, Spleen	BAT, Epi, Kidney, Lung Spleen
	Cys-438	NR	Brain, Kidney, Liver, Spleen	BAT, Brain, Liver, Spleen
	Cys-487	ND (Activating) [62]	BAT, Brain, Lung, Spleen	Bat, Brain, Epi, Kidney, Lung, Spleen
HCK	Cys-238	NR	Spleen	Not detected
	Cys-400	NR	Spleen	Not detected
	Cys-487	NR	Lung, Spleen	Lung, Spleen
	Not specified	ND (Activating) [100]
LCK	Cys-400	NR	Spleen	Spleen
	Cys-487 & Cys-498	ND (Activating) [101]	Not detected	Not detected

Adapted and modified from Table 1 of ref [16]. ¹Sequece numbers in this table are from chicken Src for consistency. ²Oximouse tissue abbreviations (BAT, brown adipose tissue; epi, epididymal fat; subQ, subcutaneous fat; skm, skeletal muscle). NR “None Reported” designates a specific cysteine residue where oxidation has not been addressed previously. ND “Not Determined” designates a cysteine where the referenced study does not determine the modification. A graphical representation of the Oximouse percent oxidations along with sequence conservation can be found in Fig. 3, Fig. 5.