Molecular level understanding of the functionality of PDZ3 variants via advanced all-atom simulations and dynamic residue network analyses

The third PDZ domain of PSD-95 (PDZ3) constitutes a common model to study single domain allostery without significant structural changes. In PDZ3, H372 directly connected to the binding site and G330 holding an off-binding-site position, were designated to assess the effect of mutations on binding selectivity. It has been observed that the H372A and G330T-H372A mutations change ligand preferences from class I (T/S amino acid at position -2 of the ligand) to class II (hydrophobic amino acid at the same position). Alternatively, the G330T single mutation leads to the recognition of both ligand classes. We have performed a series of molecular dynamics (MD) simulations for previously mentioned PDZ3 variants in the absence and presence of both types of ligands. With the combination of free energy difference calculations and a detailed analysis of MD trajectories, binding behavior of PDZ3 mutants, as well as their effects on ligand selection and binding affinities are explained. To scrutinize the residue-by-residue interaction we employ graph theory, and we assess dynamical community composition by using Girvan-Newman algorithm. We find that the highly charged and distal N-terminus share the same community with the ligand in the functional complexes. N- and C-termini of PDZ3 share communities, and α3 acts as a hub for the whole protein by sustaining the communication with all structural segments. Thus, ligand binding fate in PDZ3 is traced to the population of community compositions extracted from dynamics despite the lack of significant conformational changes

Structural dynamics of von hippel-lindau tumor supressor protein

VON HIPPEL-LINDAU TÜMÖR BASKILAYICI PROTEİNİNİN YAPISAL DİNAMİĞİ Böbrek kanseri dahil birçok organda selim ve habis tümör oluşumuyla nitelenen kalıtsal otozomal dominant bir hastalık olan von Hippel-Lindau (VHL) kanser sendromu, von Hippel-Lindau tümör baskılayıcı proteini (pVHL) üzerindeki mutasyonlarla ilişkilidir. Elongin C-Elongin B bileşiğine bağlı bulunan pVHL üzerindeki mutasyon, hipoksiyle indüklenebilir faktör (HIF) ve vasküler endotelyal büyüme faktörü (VEGF) üretimini aktive ederek tümör oluşumuna yol açar. Bu çalışmada, bilgisayar ortamında tasarlanan Elongin C ve HIF zincirlerine bağlı pVHL yapısı, Moleküler Dinamik (MD) simülasyon metodu kullanılarak simüle edilmiş ve elde edilen yapılardan veri kümeleme metodu ile temsilci yapılar seçilerek yapısal dinamik analizlerinde kullanılmıştır. Bileşiğin yaban tipi ve tasarlanmış Y98N, Y98N-G123F ve Y98N-D179N mutant yapılarının dinamiği, proteinlerde normal modlara dayalı üç boyutlu harmonik titreşimsel analiz yaparak kollektif hareketlerinin yönlerini ve boyutlarını tahmin eden Eşyönsüz Ağyapı Modeli (ANM) ile karşılaştırmalı olarak incelenmiştir. Rezidülerin dalgalanma yönleri, özellikle hareket boyutları en düşük olan ve dinamik olarak önemli dayanak bölgelerinin etrafında değişiklik göstermektedir. Bu da, proteinlerde dayanak bölgelerinin dinamik davranış biçiminin işlevle ilişkili olduğunu ispatlamaktadır. ANM ile hesaplanan en kooperatif işlevsel hareket modlarında protein rezidülerindeki dalgalanma yönleri ve dalgalanmalar arasındaki korelasyonların analizi sonucunda, yaban tipi dinamik davranış biçiminin Y98N mutant yapınınkine kıyasla Y98N-G123F mutant yapısınınkine benzer olduğu gösterilmiştir. Y98N-D179N çiftli mutant yapısında ise, Y98N mutantından sapmalar ve yaban tipine benzerlikler daha çok dalgalanmalar arasındaki korelasyonlarlarda belirgindir. Bu sonuçlar, hastalıkla alakalı proteine eklenen ikinci mutasyonlarla yaban tipi dinamiği ve işlevinin geri kazanıldığını göstermektedir. Genel olarak bu çalışma, pVHL sistemindeki moleküler etkileşimlerin ve evrimsel optimizasyonun yapısal dinamik temellerini açıklayarak VHL hastalığına yönelik ilaç tasarımı çalışmalarına yardımcı olacaktır. ABSTRACT STRUCTURAL DYNAMICS OF VON HIPPEL-LINDAU TUMOR SUPPRESSOR PROTEIN The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations on the von Hippel-Lindau tumor suppressor protein (pVHL), which activates the production of the hypoxia inducible factor (HIF) and the vascular endothelial growth factor (VEGF) leading to tumor growth. In this work, the mutant structures of the pVHL bound to Elongin C and HIF which were built in silico were relaxed by Molecular Dynamics (MD) simulations and the representative members of the MD trajectories generated by clustering were submitted to further computational structural dynamics analysis. The dynamics of wild-type, tumor-related Y98N mutant, and Y98N-G123F and Y98N-D179N double mutant complex structures were assessed comparatively utilizing the Anisotropic Network Model (ANM). The directions of fluctuations differ mainly around the important hinge regions in the minima of the most cooperative mode shapes, justifying that the dynamic nature of hinges should correlate with the functioning of proteins. The analyses of orientation of fluctuations and correlations between fluctuations in the functional modes of ANM indicate that the dynamic behavior of the Y98N-G123F double mutant is similar to that of the wild-type compared to the Y98N mutant which is known to be associated with the VHL disease. However, in the Y98N-D179N double mutant structure, deviation from Y98N mutant and accordance with wild-type is observed mostly in the correlations between fluctuations. These results indicate a regain of wild-type dynamics, and thus wild-type function, with the addition of the second mutations to the disease-related variant. Overall, this study would help in the drug design studies for the VHL disease by explaining molecular recognition and evolutionary optimization in the pVHL system from a structural dynamics perspective

Dynamic community composition unravels allosteric communication in pdz3

The third domain of PSD-95 (PDZ3) is a model for investigating allosteric communication in protein and ligand interactions. While motifs contributing to its binding specificity have been scrutinized, a conformational dynamical basis is yet to be established. Despite the miniscule structural changes due to point mutants, the observed significant binding affinity differences have previously been assessed with a focus on two α-helices located at the binding groove (α2) and the C-terminus (α3). Here, we employ a new computational approach to develop a generalized view on the molecular basis of PDZ3 binding selectivity and interaction communication for a set of point mutants of the protein (G330T, H372A, G330T-H372A) and its ligand (CRIPT, named L1, and its T-2F variant, L2) along with the wild type (WT). To analyze the dynamical aspects hidden in the conformations that are produced by molecular dynamics simulations, we utilize variations in community composition calculated based on the betweenness centrality measure from graph theory. We find that the highly charged N-terminus, which is located far from the ligand, has the propensity to share the same community with the ligand in the biologically functional complexes, indicating a distal segment might mediate the binding dynamics. N- and C-termini of PDZ3 share communities, and α3 acts as a hub for the whole protein by sustaining the communication with all structural segments, albeit being a trait not unique to the functional complexes. Moreover, α2 which lines the binding cavity frequently parts communities with the ligand and is not a controller of the binding but is rather a slave to the overall dynamics coordinated by the N-terminus. Thus, ligand binding fate in PDZ3 is traced to the population of community compositions extracted from dynamics despite the lack of significant conformational changes

N-terminus of the third PDZ domain of PSD-95 orchestrates allosteric communication for selective ligand binding

PDZ domains constitute common models to study single-domain allostery without significant structural changes. The third PDZ domain of PSD-95 (PDZ3) is known to have selective structural features that confer unique modulatory roles to this unit. In this model system, two residues, H372 directly connected to the binding site and G330 holding an off-binding-site position, were designated to assess the effect of mutations on binding selectivity. It has been observed that the H372A and G330T-H372A mutations change ligand preferences from class I (T/S amino acid at position -2 of the ligand) to class II (hydrophobic amino acid at the same position). Alternatively, the G330T single mutation leads to the recognition of both ligand classes. We have performed a series of molecular dynamics (MD) simulations for wild-type, H372A, and G330T single mutants and a double mutant of PDZ3 in the absence and presence of both types of ligands. With the combination of free-energy difference calculations and a detailed analysis of MD trajectories, "class switching"and "class bridging"behavior of PDZ3 mutants, as well as their effects on ligand selection and binding affinities are explained. We show that the dynamics of the charged N-terminus plays a fundamental role in determining the binding preferences in PDZ3 by altering the electrostatic energy. These findings are corroborated by simulations on N-terminus-truncated versions of these systems. The dynamical allostery orchestrated by the N-terminus offers a fresh perspective to the study of communication pathways in proteins

Kinetic barrier to enzyme inhibition is manipulated by dynamical local interactions in E. coli DHFR

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model protein for understanding enzyme dynamics. DHFR’s crucial role in folate synthesis renders it an ideal candidate to understand protein function and protein evolution mechanisms. In this study, to understand how a newly proposed DHFR inhibitor, 4′-deoxy methyl trimethoprim (4′-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior performance over that of trimethoprim (TMP). To elucidate the inhibition mechanism of 4′-DTMP, we first confirmed, both computationally and experimentally, that the relative binding free energy cost for the mutation of TMP and 4′-DTMP is the same, pointing the origin of the characteristic differences to be kinetic rather than thermodynamic. We then employed an interaction-based analysis by focusing first on the active site and then on the whole enzyme. We confirmed that the polar modification in 4′-DTMP induces additional local interactions with the enzyme, particularly, the M20 loop. These changes are propagated to the whole enzyme as shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support our analysis with network-based community analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor