Intramolecular binding of a proximal PPII helix to an SH3 domain in the fusion protein SH3Hck : PPIIhGAP.
Gmeiner WH, Xu I, Horita DA, Smithgall TE, Engen JR, Smith DL, Byrd RA.
Cell Biochem Biophys. 2001;35(2):115-26.

SH3 domains are a conserved feature of many nonreceptor protein tyrosine kinases, such as Hck, and often function in substrate recruitment and regulation of kinase activity. SH3 domains modulate kinase activity by binding to polyproline helices (PPII helix) either intramolecularly or in target proteins. The preponderance of bimolecular and distal interactions between SH3 domains and PPII helices led us to investigate whether proximal placement of a PPII helix relative to an SH3 domain would result in tight, intramolecular binding. We have fused the PPII helix region of human GAP to the C-terminus of Hck SH3 and expressed the recombinant fusion protein in Escherichia coli. The fusion protein, SH3Hck : PPIIhGAP, folded spontaneously into a structure in which the PPII helix was bound intramolecularly to the hydrophobic crevice of the SH3 domain. The SH3Hck : PPIIhGAP fusion protein is useful for investigating SH3: PPII helix interactions, for studying concepts in protein folding and design, and may represent a protein structural motif that is widely distributed in nature.

Pubmed: 11892787

Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase.
Dorey K, Engen JR, Kretzschmar J, Wilm M, Neubauer G, Schindler T, Superti-Furga G.
Oncogene. 2001 Dec 6;20(56):8075-84.

c-Abl is a nuclear and cytoplasmic tyrosine kinase involved in a variety of cellular growth and differentiation processes. In contrast to its oncogenic counterparts, like BCR-Abl, c-Abl is not constitutively tyrosine phosphorylated and its catalytic activity is very low. Here we report tyrosine phosphorylation of endogenous c-Abl and a concomitant increase in catalytic activity. Using Abl -/- cells reconstituted with mutated c-Abl forms, we show that phosphorylation and activity depend on Tyr412 in the activation loop. Tyr412 is also required for stimulation by PDGF or by cotransfection of active Src. Phosphorylation of Tyr412 can occur autocatalytically by a trans-mechanism and cause activation of otherwise inactive c-Abl, suggesting a positive feedback loop on c-Abl activity. In the recent structure of the Abl catalytic domain bound to the STI-571 inhibitor, unphosphorylated Tyr412 in the activation loop points inward and appears to interfere with catalysis. We mutated residues involved in stabilizing this inhibited form of the activation loop and in positioning Tyr412. These mutations resulted in tyrosine phosphorylation and activation of c-Abl, as if relieving c-Abl from inhibition. Tyr412 is therefore necessary both for activity and for regulation of c-Abl, by stabilizing the inactive or the active conformation of the enzyme in a phosphorylation-dependent manner.
Pubmed: 11781820

Investigating protein structure and dynamics by hydrogen exchange MS.

Engen JR, Smith DL.
Anal Chem. 2001 May 1;73(9):256A-265A

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Pubmed: 11354508

Investigating the higher order structure of proteins. Hydrogen exchange, proteolytic fragmentation, and mass spectrometry.
Engen JR, Smith DL.
Methods Mol Biol. 2000;146:95-112.

It has been apparent for some years that the structures of proteins are dynamic rather than static. For some proteins, dynamics is essential to function (e.g., refs. 1–7). These structural changes have been detected for more than 30 yr by observing hydrogen exchange between peptide amide hydrogens and solvent containing the hydrogen isotopes tritium or deuterium (Refs. 8-10). Although tritium is no longer used extensively for this purpose, deuterium is widely used in hydrogen exchange studies, especially in multidimensional nuclear magnetic resonance (NMR), in which amide hydrogen signals disappear on deuteration. Since deuterium weighs 1 Dalton more than protium, hydrogen exchange in proteins can also be detected by mass spectrometry. This approach is complementary to NMR in some respects and clearly advantageous in others.
 Pubmed: 10948498

Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2.
Engen JR, Gmeiner WH, Smithgall TE, Smith DL.
Biochemistry. 1999 Jul 13;38(28):8926-35.

Src-homology-2 domains are small, 100 amino acid protein modules that are present in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide binding decreases protein motions in the pico- to nanosecond, and perhaps slower, time range. We suggest that amide hydrogen exchange and mass spectrometry may be useful for detecting changes in protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of the dynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchange rates were determined by exposing free and bound SH2 to D(2)O, fragmenting the SH2 with pepsin, and determining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in the exchange of the most rapidly exchanging amide hydrogens, which was detected following acid quench and analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded form of SH2 is an important process along the entire SH2 backbone.
Pubmed: 10413466

Comparison of SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation.
Engen JR, Smithgall TE, Gmeiner WH, Smith DL.
J Mol Biol. 1999 Apr 2;287(3):645-56.

Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3+2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater flexibility of SH3 when it is part of SH(3+2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3+2) construct, suggesting a functional significance for this unfolding. The SH2 domain displayed relatively smaller changes in flexibility when part of the SH(3+2) construct. These results suggest that the domains influence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains.
Pubmed: 10092465

Identification and localization of slow, natural, cooperative unfolding in the hematopoietic cell kinase SH3 domain by amide hydrogen exchange and mass spectrometry.
Engen JR, Smithgall TE, Gmeiner WH, Smith DL.
Biochemistry. 1997 Nov 25;36(47):14384-91.

Protein unfolding on a fast time scale (milliseconds-minutes) has been widely reported, but slower unfolding events (10 min-hours) have received less attention. Amide hydrogen exchange (HX) and mass spectrometry (MS) were used to investigate the unfolding dynamics of the hematopoietic cell kinase (Hck) SH3 domain. Analysis of mass spectra after deuterium exchange into intact Hck SH3 indicates a cooperative unfolding event involving 24-61% of the domain and occurring with a half-life of approximately 20 min under physiological conditions. To identify the unfolding region, SH3 was incubated in D2O and proteolytically fragmented into peptides that were analyzed by mass spectrometry. Correlation of HX rates and isotope patterns reveals cooperative unfolding in several regions, including the C-terminal half of the RT-loop and a beta-sheet flanking the binding site. Binding of a prolyl-rich segment from the HIV Nef protein slows unfolding by a factor of 3. Further analysis yields a KD of 25 microM for the Nef peptide. These results demonstrate that an inherent flexibility in the SH3 domain may assist interconversion of the closed, intramolecularly ligated state and the open, active state of Src family kinases. Furthermore, this type of previously undetectable, slow unfolding process may provide the basis for new mechanisms in which kinetics of local unfolding combines with thermodynamics to regulate enzymatic activity. The combination of hydrogen exchange and mass spectrometry appears to be the only general method capable of examining these slow unfolding processes.
Pubmed: 9398156