系统比较氨基酸描述子在多肽定量构效关系中的应用

氨基酸描述子是采用多元统计方法从大量氨基酸性质参数中提取得到的少数关键信息成分,目前已被广泛应用于多肽生物活性预测及蛋白质功能判别等领域。鉴于近年来氨基酸描述子种类的极速扩增,收集了目前国内外发表的27种氨基酸描述子,并将其用于8组经典多肽集的结构表征及定量构效关系研究。通过系统比较这些描述子对同一肽集与不同肽集的统计建模结果,我们认为物理化学描述子的建模效果优于拓扑描述子,拓扑描述子的建模效果优于三维结构描述子,且已有诸多氨基酸描述子已经达到性能限度,如未考虑肽链内部各氨基酸残基的交互影响,肽配基与相应靶标蛋白的相互结合,因此不再建议按照传统思路进一步提出新型描述子种类。     

Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.     

Solution structure of the human BTK SH3 domain complexed with a proline-rich peptide from p120cbl

X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B cell differentiation which incapacitates antibody production in XLA patients leading to, sometimes lethal, bacterial infections. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in one patient family. To understand the role of BTK in B cell development, we have determined the solution structure of BTK SH3 domain complexed with a proline-rich peptide from the protein product of c-cbl protooncogene (p120^cbl). Like other SH3 domains, BTK SH3 domain consists of five -strands packed in two -sheets forming a -barrel-like structure. The rmsd calculated from the averaged coordinates for the BTK SH3 domain residues 218–271 and the p120^cbl peptide residues 6–12 of the complex was 0.87 Å (±0.16 Å) for the backbone heavy atoms (N, C, and C) and 1.64 Å (±0.16 Å) for all heavy atoms. Based on chemical shift changes and inter-molecular NOEs, we have found that the residues located in the RT loop, n-Src loop and helix-like loop between 4 and 5 of BTK SH3 domain are involved in ligand binding. We have also determined that the proline-rich peptide from p120^cbl binds to BTK SH3 domain in a class I orientation. These results correlate well with our earlier observation that the truncated BTK SH3 domain (deletion of 4, 5 and the helix-like loop) exhibits weaker affinity for the p120^cbl peptide. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context and hence the weaker binding. These results delineate the importance of the C-terminus in the binding of SH3 domains and also indicate that improper folding and the altered binding behavior of mutant BTK SH3 domain likely lead to XLA.     

Linear combinations of docking affinities explain quantitative differences in RTK signaling

Receptor tyrosine kinases (RTKs) process extracellular cues by activating a broad array of signaling proteins. Paradoxically, they often use the same proteins to elicit diverse and even opposing phenotypic responses. Binary, ‘on–off’ wiring diagrams are therefore inadequate to explain their differences. Here, we show that when six diverse RTKs are placed in the same cellular background, they activate many of the same proteins, but to different quantitative degrees. Additionally, we find that the relative phosphorylation levels of upstream signaling proteins can be accurately predicted using linear models that rely on combinations of receptor‐docking affinities and that the docking sites for phosphoinositide 3‐kinase (PI3K) and Shc1 provide much of the predictive information. In contrast, we find that the phosphorylation levels of downstream proteins cannot be predicted using linear models. Taken together, these results show that information processing by RTKs can be segmented into discrete upstream and downstream steps, suggesting that the challenging task of constructing mathematical models of RTK signaling can be parsed into separate and more manageable layers.     

Enhanced process understanding and multivariate prediction of the relationship between cell culture process and monoclonal antibody quality

This work investigates the insights and understanding which can be deduced from predictive process models for the product quality of a monoclonal antibody based on designed high‐throughput cell culture experiments performed at milliliter (ambr‐15^®) scale. The investigated process conditions include various media supplements as well as pH and temperature shifts applied during the process. First, principal component analysis (PCA) is used to show the strong correlation characteristics among the product quality attributes including aggregates, fragments, charge variants, and glycans. Then, partial least square regression (PLS1 and PLS2) is applied to predict the product quality variables based on process information (one by one or simultaneously). The comparison of those two modeling techniques shows that a single (PLS2) model is capable of revealing the interrelationship of the process characteristics to the large set product quality variables. In order to show the dynamic evolution of the process predictability separate models are defined at different time points showing that several product quality attributes are mainly driven by the media composition and, hence, can be decently predicted from early on in the process, while others are strongly affected by process parameter changes during the process. Finally, by coupling the PLS2 models with a genetic algorithm first the model performance can be further improved and, most importantly, the interpretation of the large‐dimensioned process–product‐interrelationship can be significantly simplified. The generally applicable toolset presented in this case study provides a solid basis for decision making and process optimization throughout process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1368–1380, 2017     

P120-Ras GTPase activating protein (RasGAP): A multi-interacting protein in downstream signaling

p120-RasGAP (Ras GTPase activating protein) plays a key role in the regulation of Ras-GTP bound by promoting GTP hydrolysis via its C-terminal catalytic domain. The p120-RasGAP N-terminal part contains two SH2, SH3, PH (pleckstrin homology) and CaLB/C2 (calcium-dependent phospholipid-binding domain) domains. These protein domains allow various functions, such as anti-/pro-apoptosis, proliferation and also cell migration depending of their distinct partners. The p120-RasGAP domain participates in protein–protein interactions with Akt, Aurora or RhoGAP to regulate functions described bellow. Here, we summarize, in angiogenesis and cancer, the various functional roles played by p120-RasGAP domains and their effector partners in downstream signaling.     

Crystal structure of the SH3 domain of betaPIX in complex with a high affinity peptide from PAK2

The p21-activated kinases (PAKs) are important effector proteins of the small GTPases Cdc42 and Rac and control cytoskeletal rearrangements and cell proliferation. The direct interaction of PAKs with guanine nucleotide exchange factors from the PIX/Cool family, which is responsible for the localization of PAK kinases to focal complexes in the cell, is mediated by a 24-residue peptide segment in PAKs and an N-terminal src homology 3 (SH3) domain in PIX/Cool. The SH3-binding segment of PAK contains the atypical consensus-binding motif PxxxPR, which is required for unusually high affinity binding. In order to understand the structural basis for the high affinity and specificity of the PIX-PAK interaction, we solved crystal structures for the N-terminal SH3 domain of betaPIX and for the complex of the atypical binding segment of PAK2 with the N-terminal SH3 domain of betaPIX at 0.92 A and 1.3A resolution, respectively. The asymmetric unit of the crystal contains two SH3 domains and two peptide ligands. The bound peptide adopts a conformation that allows for intimate contacts with three grooves on the surface of the SH3 domain that lie between the n-Src and RT-loops. Most notably, the arginine residue of the PxxxPR motif forms a salt-bridge and is tightly coordinated by a number of residues in the SH3 domain. This arginine-specific interaction appears to be the key determinant for the high affinity binding of PAK peptides. Furthermore, C-terminal residues of the peptide engage in additional interactions with the surface of the RT-loop, which significantly increases binding specificity. Compared to a recent NMR structure of a similar complex, our crystal structure reveals an alternate binding mode. Finally, we compare our crystal structure with the recently published betaPIX/Cbl-b complex structure, and suggest the existence of a molecular switch.     

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain

To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.