Cooperative subunit dynamics modulate p97 function.

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.

LRPs in WNT Signalling.

The WNT/ß-catenin signalling pathway is a rich and complex network of cellular proteins that orchestrates diverse short-range cell-to-cell communication in metazoans and is essential for both embryonic development and adult homeostasis. Due to its fundamental importance in controlling cell behaviour at multiple levels, its deregulation is associated with a wide range of diseases in humans and identification of drugs targeting the pathway has attracted strong interest in the pharmaceutical sector. Transduction of WNT signals across the plasma membrane of cells involves a staggering degree of complexity and variety with respect to ligand-receptor, receptor-receptor and receptor-co-receptor interactions (Niehrs, Nat Rev Mol Cell Biol 13:767-779, 2012). Although the low-density-lipoprotein-receptor-related-protein (LRP) family is best known for its role in binding and endocytosis of lipoproteins, specific members appear to have additional roles in cellular communication. Indeed, for WNT/ß-catenin signalling one apparently universal requirement is the presence of either LRP5 or LRP6 in combination with one of the ten Frizzled (FZD) WNT receptors (FZD1-10). In the 20 years since their discovery as WNT/FZD co-receptors, research on the LRP family has contributed greatly to our understanding of WNT signalling and LRPs have emerged as central players in WNT/ß-catenin signalling. LRP5/6 are highly similar and represent the least redundant class of WNT receptor that transduce WNT/ß-catenin signalling from a wide range of different WNT and FZD subtypes. This apparent simplicity however belies the complex arrangement of binding sites in the extracellular domain (ECD) of LRP5/6, which regulate interaction not only with WNTs but also with several inhibitors of WNT signalling. This chapter provides a historical overview, chronologically charting this remarkable progress in the field during the last 20 years of research on LRPs and their role in WNT/-catenin signalling. A more focused overview of the structural, functional and mechanistic aspects of LRP biology is also provided, together with the implications this has for pharmacological targeting of this notoriously intractable pathway.

Disruption of protein function by pathogenic mutations: common and uncommon mechanisms 1.

Mutations in protein-coding regions underlie almost all Mendelian disorders, drive tumorigenesis, and contribute to susceptibility to common diseases. Despite the great diversity of diseases that are caused by coding mutations, the cellular processes that affect, and are affected by, pathogenic variants at the molecular level are fundamentally conserved. Experimental and computational approaches have revealed that a substantial fraction of disease mutations are not simple loss-of-function alleles. Rather, these pathogenic variants disrupt protein function in more subtle ways by tuning protein folding pathways, altering subcellular trafficking, interrupting signaling cascades, and rewiring highly connected interaction networks. Focusing mainly on Mendelian disorders, this review discusses the common mechanisms by which deleterious mutations disrupt protein function and how these disruptions can be exploited in the development of novel therapies.

Structural Determinants of the Prion Protein N-Terminus and Its Adducts with Copper Ions.

The N-terminus of the prion protein is a large intrinsically disordered region encompassing approximately 125 amino acids. In this paper, we review its structural and functional properties, with a particular emphasis on its binding to copper ions. The latter is exploited by the region's conformational flexibility to yield a variety of biological functions. Disease-linked mutations and proteolytic processing of the protein can impact its copper-binding properties, with important structural and functional implications, both in health and disease progression.

Mapping genotype-phenotype associations of nsSNPs in coiled-coil oligomerization domains of the human proteome.

We assessed the impact of disease mutations (DMs) versus polymorphisms (PYs) in coiled-coil (CC) domains in UniProt by modeling the structural and functional impact of variants in silico with the CC prediction program Multicoil. The structural impact of variants was evaluated with respect to three main metrics: the oligomerization score-to determine whether the variant is stabilizing or destabilizing-the oligomerization state, and the register-specific score. The functional impact was queried indirectly in several ways. First, we examined marginally stable CCs that were either stabilized or destabilized by the variant. Second, we looked for variants that altered the register of the wild-type CC near wild-type irregularities of likely functional importance, such as skips and stammers. Third, we searched for variants that altered the oligomerization state of the CC. DMs tended to be more destabilizing than PYs; but interestingly, PYs were more frequently associated with predicted changes in the oligomerization state. The functional impact was also queried by testing the association of CC variants with multiple phenotypes, that is, pleiotropy. Mutations in CC regions of proteins cause 155 different phenotypes and are more frequently associated with pleiotropy than proteins in general. Importantly, the CC region itself often encodes the pleiotropy.

Enhanced Cytosolic Ca2+ Activation Underlies a Common Defect of Central Domain Cardiac Ryanodine Receptor Mutations Linked to Arrhythmias.

Recent three-dimensional structural studies reveal that the central domain of ryanodine receptor (RyR) serves as a transducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to amino acid residues 3778-4201) that contains a number of cardiac RyR (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and atrial fibrillation (AF). However, the functional consequences of these central domain RyR2 mutations are not well understood. To gain insights into the impact of the mutation and the role of the central domain in channel function, we generated and characterized eight disease-associated RyR2 mutations in the central domain. We found that all eight central domain RyR2 mutations enhanced the Ca2+-dependent activation of [3H]ryanodine binding, increased cytosolic Ca2+-induced fractional Ca2+ release, and reduced the activation and termination thresholds for spontaneous Ca2+ release in HEK293 cells. We also showed that racemic carvedilol and the non-beta-blocking carvedilol enantiomer, (R)-carvedilol, suppressed spontaneous Ca2+ oscillations in HEK293 cells expressing the central domain RyR2 mutations associated with CPVT and AF. These data indicate that the central domain is an important determinant of cytosolic Ca2+ activation of RyR2. These results also suggest that altered cytosolic Ca2+ activation of RyR2 represents a common defect of RyR2 mutations associated with CPVT and AF, which could potentially be suppressed by carvedilol or (R)-carvedilol.

Cell lipid metabolism modulators 2-bromopalmitate, D609, monensin, U18666A and probucol shift discoidal HDL formation to the smaller-sized particles: implications for the mechanism of HDL assembly.

ATP-binding cassette transporter A1 (ABCA1) mediates formation of disc-shaped high-density lipoprotein (HDL) from cell lipid and lipid-free apolipoprotein A-I (apo A-I). Discoidal HDL particles are heterogeneous in physicochemical characteristics for reasons that are understood incompletely. Discoidal lipoprotein particles similar in characteristics and heterogeneity to cell-formed discoidal HDL can be reconstituted from purified lipids and apo A-I by cell-free, physicochemical methods. The heterogeneity of reconstituted HDL (rHDL) is sensitive to the lipid composition of the starting lipid/apo A-I mixture. To determine whether the heterogeneity of cell-formed HDL is similarly sensitive to changes in cell lipids, we investigated four compounds that have well-established effects on cell lipid metabolism and ABCA1-mediated cell cholesterol efflux. 2-Bromopalmitate, D609, monensin and U18666A decreased formation of the larger-sized, but dramatically increased formation of the smaller-sized HDL. 2-Bromopalmitate did not appear to affect ABCA1 activity, subcellular localization or oligomerization, but induced dissolution of the cholesterol-phospholipid complexes in the plasma membrane. Arachidonic and linoleic acids shifted HDL formation to the smaller-sized species. Tangier disease mutations and inhibitors of ABCA1 activity wheat germ agglutinin and AG 490 reduced formation of both larger-sized and smaller-sized HDL. The effect of probucol was similar to the effect of 2-bromopalmitate. Taking rHDL formation as a paradigm, we propose that ABCA1 mutations and activity inhibitors reduce the amount of cell lipid available for HDL formation, and the compounds in the 2-bromopalmitate group and the polyunsaturated fatty acids change cell lipid composition from one that favors formation of the larger-sized HDL particles to one that favors formation of the smaller-sized species.

Structural consequences of mutations associated with idiopathic restrictive cardiomyopathy.

Idiopathic restrictive cardiomyopathy (RCM, MIM# 115210) is the least common type of cardiomyopathies, often of genetic origin. Recently we described a spectrum of variants-classified as pathogenic, likely pathogenic and variants of unknown significance-in 24 patients suffering from idiopathic RCM. Pathogenic variants, detected in half of the RCM cases, were found in sarcomeric and cytoskeletal genes that have a predominant role in the development of RCM. Here we have analyzed the structural consequences of these missense variants and predicted their effect on the function of three large groups of domains: intrinsically disordered regions (IDRs), fibronectin-type III (FnIII) domains, and immunoglobulin-like (Ig) domains. Our findings indicate that pathogenic mutations are likely to disrupt interdomain interfaces, interfere with protein interactions, and affect protein stability, potentially destabilizing the multi-domain architecture of myofibrils and leading to myocardial stiffness in patients with idiopathic RCM.

Cell-free expression of the APP transmembrane fragments with Alzheimer's disease mutations using algal amino acid mixture for structural NMR studies.

Structural investigations need ready supply of the isotope labeled proteins with inserted mutations n the quantities sufficient for the heteronuclear NMR. Though cell-free expression system has been widely used in the past years, high startup cost and complex compound composition prevent many researches from the developing this technique, especially for membrane protein production. Here we demonstrate the utility of a robust, cost-optimized cell-free expression technique for production of the physiologically important transmembrane fragment of amyloid precursor protein, APP686-726, containing Alzheimer's disease mutations in the juxtamembrane (E693G, Arctic form) and the transmembrane parts (V717G, London form, or L723P, Australian form). The protein cost was optimized by varying the FM/RM ratio as well as the amino acid concentration. We obtained the wild-type and mutant transmembrane fragments in the pellet mode of continuous exchange cell-free system consuming only commercial algal mixture of the (13)C,(15)N-labeled amino acids. Scaling up analytical tests, we achieved milligram quantity yields of isotope labeled wild-type and mutant APP686-726 for structural studies by high resolution NMR spectroscopy in membrane mimicking environment. The described approach has from 5 to 23-fold cost advantage over the bacterial expression methods described earlier and 1.5 times exceeds our previous result obtained with the longer APP671-726WT fragment.

Decrease in catalytic capacity of γ-secretase can facilitate pathogenesis in sporadic and Familial Alzheimer's disease.

BACKGROUND: Alzheimer's disease can be a result of an age-induced disparity between increase in cellular metabolism of Aß peptides and decrease in maximal activity of a membrane-embedded protease γ-secretase. RESULTS: We compared activity of WT γ-secretase with the activity of 6 FAD mutants in its presenilin-1 component and 5 FAD mutants in Aß-part of its APP substrate (Familial Alzheimer's disease). All 11 FAD mutations show linear correlation between the decrease in maximal activity and the clinically observed age-of-onset and age-of-death. Biphasic-inhibitors showed that a higher ratio between physiological Aß-production and the maximal activity of γ-secretase can be observed in cells that can facilitate pathogenic changes in Aß-products. For example, Aß production in cells with WT γ-secretase is at 11% of its maximal activity, with delta-exon-9 mutant at 26%, while with M139V mutant is at 28% of the maximal activity. In the same conditions, G384A mutant is fully saturated and at its maximal activity. Similarly, Aß production in cells with γ-secretase complex carrying Aph1AL component is 12% of its maximal activity, while in cells with Aph1B complex is 26% of its maximal activity. Similar to the cell-based studies, clinical studies of biphasic dose-response in plasma samples of 54 healthy individuals showed variable ratios between physiological Aß production and the maximal activity of γ-secretase. CONCLUSIONS: The increase in the ratio between physiological Aß production and maximal activity of γ-secretase can be an early sign of pathogenic processes in enzyme-based, cell-based, and clinical studies of sporadic and Familiar Alzheimer's disease.

Crystallographic insights into sodium-channel modulation by the ß4 subunit.

Voltage-gated sodium (Nav) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, ß-subunits modify Nav channel function and cause debilitating disorders when mutated. While investigating whether ß-subunits also influence ligand interactions, we found that ß4 dramatically alters toxin binding to Nav1.2. To explore these observations further, we solved the crystal structure of the extracellular ß4 domain and identified (58)Cys as an exposed residue that, when mutated, eliminates the influence of ß4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of ß4. Disrupting this bridge by introducing a ß1 mutation implicated in epilepsy repositions the (58)Cys-containing loop and disrupts ß4 modulation of Nav1.2. Overall, the principles emerging from this work (i) help explain tissue-dependent variations in Nav channel pharmacology; (ii) enable the mechanistic interpretation of ß-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant ß-subunit behavior.

On human disease-causing amino acid variants: statistical study of sequence and structural patterns.

Statistical analysis was carried out on large set of naturally occurring human amino acid variations, and it was demonstrated that there is a preference for some amino acid substitutions to be associated with diseases. At an amino acid sequence level, it was shown that the disease-causing variants frequently involve drastic changes in amino acid physicochemical properties of proteins such as charge, hydrophobicity, and geometry. Structural analysis of variants involved in diseases and being frequently observed in human population showed similar trends: disease-causing variants tend to cause more changes in hydrogen bond network and salt bridges as compared with harmless amino acid mutations. Analysis of thermodynamics data reported in the literature, both experimental and computational, indicated that disease-causing variants tend to destabilize proteins and their interactions, which prompted us to investigate the effects of amino acid mutations on large databases of experimentally measured energy changes in unrelated proteins. Although the experimental datasets were linked neither to diseases nor exclusory to human proteins, the observed trends were the same: amino acid mutations tend to destabilize proteins and their interactions. Having in mind that structural and thermodynamics properties are interrelated, it is pointed out that any large change in any of them is anticipated to cause a disease.

PACS-1 variant protein is aberrantly localized in Caenorhabditis elegans model of PACS1/PACS2 syndromes.

PACS (phosphofurin acidic cluster sorting) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of Caenorhabditis elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 colocalize to somatic cytoplasm of many types of cells and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

PACS-1 variant protein is aberrantly localized in C. elegans model of PACS1/PACS2 syndromes.

PACS (Phosphofurin Acidic Cluster Sorting Protein) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the effects of syndromic variants on function in vivo remains unknown. Here, we report the expression pattern of C. elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 co-localize to somatic cytoplasm of many types of cells, and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

Disease-Associated Mutations in Tau Encode for Changes in Aggregate Structure Conformation.

The accumulation of tau fibrils is associated with neurodegenerative diseases, which are collectively termed tauopathies. Cryo-EM studies have shown that the packed fibril core of tau adopts distinct structures in different tauopathies, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy. A subset of tauopathies are linked to missense mutations in the tau protein, but it is not clear whether these mutations impact the structure of tau fibrils. To answer this question, we developed a high-throughput protein purification platform and purified a panel of 37 tau variants using the full-length 0N4R splice isoform. Each of these variants was used to create fibrils in vitro, and their relative structures were studied using a high-throughput protease sensitivity platform. We find that a subset of the disease-associated mutations form fibrils that resemble wild-type tau, while others are strikingly different. The impact of mutations on tau structure was not clearly associated with either the location of the mutation or the relative kinetics of fibril assembly, suggesting that tau mutations alter the packed core structures through a complex molecular mechanism. Together, these studies show that single-point mutations can impact the assembly of tau into fibrils, providing insight into its association with pathology and disease.

Missense mutations in CRX homeodomain cause dominant retinopathies through two distinct mechanisms.

Homeodomain transcription factors (HD TFs) are instrumental to vertebrate development. Mutations in HD TFs have been linked to human diseases, but their pathogenic mechanisms remain elusive. Here, we use Cone-Rod Homeobox (CRX) as a model to decipher the disease-causing mechanisms of two HD mutations, p.E80A and p.K88N, that produce severe dominant retinopathies. Through integrated analysis of molecular and functional evidence in vitro and in knock-in mouse models, we uncover two novel gain-of-function mechanisms: p.E80A increases CRX-mediated transactivation of canonical CRX target genes in developing photoreceptors; p.K88N alters CRX DNA-binding specificity resulting in binding at ectopic sites and severe perturbation of CRX target gene expression. Both mechanisms produce novel retinal morphological defects and hinder photoreceptor maturation distinct from loss-of-function models. This study reveals the distinct roles of E80 and K88 residues in CRX HD regulatory functions and emphasizes the importance of transcriptional precision in normal development.

Structural Insight Into Ryanodine Receptor Channelopathies.

The ryanodine receptors (RyRs) are large cation-selective ligand-gated channels that are expressed in the sarcoplasmic reticulum (SR) membrane. They mediate the controlled release of Ca2+ from SR and play an important role in many cellular processes. The mutations in RyRs are associated with several skeletal muscle and cardiac conditions, including malignant hyperthermia (MH), central core disease (CCD), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia (ARVD). Recent breakthroughs in structural biology including cryo-electron microscopy (EM) and X-ray crystallography allowed the determination of a number of near-atomic structures of RyRs, including wildtype and mutant structures as well as the structures in complex with different modulating molecules. This allows us to comprehend the physiological gating and regulatory mechanisms of RyRs and the underlying pathological mechanisms of the disease-causing mutations. In this review, based on the insights gained from the available high-resolution structures of RyRs, we address several questions: 1) what are the gating mechanisms of different RyR isoforms; 2) how RyRs are regulated by multiple channel modulators, including ions, small molecules, and regulatory proteins; 3) how do disease-causing mutations affect the structure and function of RyRs; 4) how can these structural information aid in the diagnosis of the related diseases and the development of pharmacological therapies.

A heterozygous p.S143P mutation in LMNA associates with proteasome dysfunction and enhanced autophagy-mediated degradation of mutant lamins A and C.

Lamins A and C are nuclear intermediate filament proteins that form a proteinaceous meshwork called lamina beneath the inner nuclear membrane. Mutations in the LMNA gene encoding lamins A and C cause a heterogenous group of inherited degenerative diseases known as laminopathies. Previous studies have revealed altered cell signaling pathways in lamin-mutant patient cells, but little is known about the fate of mutant lamins A and C within the cells. Here, we analyzed the turnover of lamins A and C in cells derived from a dilated cardiomyopathy patient with a heterozygous p.S143P mutation in LMNA. We found that transcriptional activation and mRNA levels of LMNA are increased in the primary patient fibroblasts, but the protein levels of lamins A and C remain equal in control and patient cells because of a meticulous interplay between autophagy and the ubiquitin-proteasome system (UPS). Both endogenous and ectopic expression of p.S143P lamins A and C cause significantly reduced activity of UPS and an accumulation of K48-ubiquitin chains in the nucleus. Furthermore, K48-ubiquitinated lamins A and C are degraded by compensatory enhanced autophagy, as shown by increased autophagosome formation and binding of lamins A and C to microtubule-associated protein 1A/1B-light chain 3. Finally, chaperone 4-PBA augmented protein degradation by restoring UPS activity as well as autophagy in the patient cells. In summary, our results suggest that the p.S143P-mutant lamins A and C have overloading and deleterious effects on protein degradation machinery and pharmacological interventions with compounds enhancing protein degradation may be beneficial for cell homeostasis.

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death.

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.

Protein Predictive Modeling and Simulation of Mutations of Presenilin-1 Familial Alzheimer's Disease on the Orthosteric Site.

Alzheimer's disease pathology is characterized by ß-amyloid plaques and neurofibrillary tangles. Amyloid precursor protein is processed by ß and γ secretase, resulting in the production of ß-amyloid peptides with a length ranging from 38 to 43 amino acids. Presenilin 1 (PS1) is the catalytic unit of γ-secretase, and more than 200 PS1 pathogenic mutations have been identified as causative for Alzheimer's disease. A complete monocrystal structure of PS1 has not been determined so far due to the presence of two flexible domains. We have developed a complete structural model of PS1 using a computational approach with structure prediction software. Missing fragments Met1-Glut72 and Ser290-Glu375 were modeled and validated by their energetic and stereochemical characteristics. Then, with the complete structure of PS1, we defined that these fragments do not have a direct effect in the structure of the pore. Next, we used our hypothetical model for the analysis of the functional effects of PS1 mutations Ala246GLu, Leu248Pro, Leu248Arg, Leu250Val, Tyr256Ser, Ala260Val, and Val261Phe, localized in the catalytic pore. For this, we used a quantum mechanics/molecular mechanics (QM/MM) hybrid method, evaluating modifications in the topology, potential surface density, and electrostatic potential map of mutated PS1 proteins. We found that each mutation exerts changes resulting in structural modifications of the active site and in the shape of the pore. We suggest this as a valid approach for functional studies of PS1 in view of the possible impact in substrate processing and for the design of targeted therapeutic strategies.