International Journal of Molecular Sciences Article Computational Investigation of the pH Dependence of Stability of Melanosome Proteins: Implication for Melanosome formation and Disease Mahesh Koirala 1,† , H. B. Mihiri Shashikala 1,†, Jacob Jeffries 1, Bohua Wu 1, Stacie K. Loftus 2, Jonathan H. Zippin 3 and Emil Alexov 1,* 1 Department of Physics, Clemson University, Clemson, SC 29634, USA; [email protected] (M.K.); [email protected] (H.B.M.S.); [email protected] (J.J.); [email protected] (B.W.) 2 Genetic Disease Research Branch, National Human Genome Research Branch, National Institutes of Health, Bethesda, MD 22066, USA; [email protected] 3 Department of Dermatology, Weill Cornell Medical College, New York, NY 10021, USA; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Intravesicular pH plays a crucial role in melanosome maturation and function. Melanoso- mal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin- synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated Citation: Koirala, M.; Shashikala, the pH-dependent stability of several melanosomal membrane proteins and compared them to H.B.M.; Jeffries, J.; Wu, B.; Loftus, the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, S.K.; Zippin, J.H.; Alexov, E. Computational Investigation of the and we also found that proteins that are negative regulators of melanosomal pH are predicted to pH Dependence of Stability of function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic Melanosome Proteins: Implication for pH optimum. We propose a competitive mechanism among positive and negative regulators that Melanosome formation and Disease. results in pH equilibrium. Our findings are consistent with previous work that demonstrated a Int. J. Mol. Sci. 2021, 22, 8273. correlation between the pH optima of stability and activity, and they are consistent with the expected https://doi.org/10.3390/ activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that ijms22158273 disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of Academic Editor: Hideya Ando multiple melanosomal proteins. Received: 7 July 2021 Keywords: pH dependence; proton transport; pH regulation; stability Accepted: 29 July 2021 Published: 31 July 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- The pH of a solution is an important characteristic for many biological processes. On iations. a molecular level, pH controls macromolecular stability and, at extreme pH (acidic or basic extremes), macromolecules unfold. Typically, for every macromolecule, there is a particular pH at which the macromolecule is the most stable and activity is maximum, termed the pH optimum [1,2]. Macromolecular interactions are also pH-dependent [3–5], and there Copyright: © 2021 by the authors. is typically a pH optimum at which the binding affinity is maximum [4]. Within a cell, Licensee MDPI, Basel, Switzerland. subcellular compartments have different pH, reflecting their function, from low pH in This article is an open access article lysosomes to high pH in peroxisomes. Thus, macromolecules tend to have a pH optimum distributed under the terms and that is ideal for the pH of the subcellular compartment where they reside [3]. Increasing conditions of the Creative Commons the scale of this idea, pH plays a crucial role for body organ function and varies from Attribution (CC BY) license (https:// very acidic in the stomach to neutral in the blood. All above examples indicate that the creativecommons.org/licenses/by/ regulation and maintenance of pH is essential for many biological phenomena. 4.0/). Int. J. Mol. Sci. 2021, 22, 8273. https://doi.org/10.3390/ijms22158273 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 13 Int. J. Mol. Sci. 2021, 22, 8273 2 of 12 from very acidic in the stomach to neutral in the blood. All above examples indicate that the regulation and maintenance of pH is essential for many biological phenomena. pH is maintained in a given cellular compartment by channels and/or pumps either pH is maintained in a given cellular compartment by channels and/or pumps either by directly trafficking H+ or by indirectly affecting the local H+ concentration. These chan- by directly trafficking H+ or by indirectly affecting the local H+ concentration. These nels and/or pumps can be termed positive (increase pH) or negative (decrease pH) regu- channels and/or pumps can be termed positive (increase pH) or negative (decrease pH) lators [6,7]. Reaching and maintaining the desired pH depends on the balance of H+ flux regulators [6,7]. Reaching and maintaining the desired pH depends on the balance of controlled by these regulators, including passive transport across the membrane (Figure H+ flux controlled by these regulators, including passive transport across the membrane 1).(Figure One would1). One expect would that expect the positive that the regulato positivers regulatorsare most active are most at acidic active pH at and acidic show pH almostand show no activity almost at no basic activity pH atsince basic their pH ro sincele is theirto increase role is pH. to increase The converse pH. The would converse be expectedwould be for expected negative for regulators, negative regulators,whereby activi wherebyty increases activity as increases the pH asrises. the At pH a rises.particu- At a + larparticular pH, the inward pH, the and inward outward and outwardflux of H flux ions ofinduced H+ ions by induced positive by and positive negative and regula- negative torsregulators becomes becomes equal and equal the andpH setp the pHoint setpointis established is established (Figure (Figure1). 1). Figure 1. Schematic representation of the induced H+ flux of positive (increase pH) and negative Figure 1. Schematic representation of the induced H+ flux of positive (increase pH) and negative (decrease pH) regulators. The vertical arrow indicates the desired pH, at which the total induced H+ (decrease pH) regulators. The vertical arrow indicates the desired pH, at which the total induced H+ flux is zero. flux is zero. Melanocytes are a specialized cell type that resides in the skin, eyes, brain, ears, heart, lungs,Melanocytes and adipose are tissue a specialized [8]. One cell of the type primary that re functionssides in the of skin, melanocytes eyes, brain, is the ears, production heart, lungs,of melanin, and adipose a polymer tissue of [8]. tyrosine One of derivativesthe primary that functions has important of melanocytes chemical is the properties produc- in tiona wide of melanin, rangeof a polymer tissues [ 9of]. tyrosine Melanin deriva is synthesizedtives that inhas a specializedimportant chemical organelle properties called the inmelanosome. a wide range Theof tissues pH of [9]. this Melanin organelle is variessynthesized during in the a specialized development organelle of the organellecalled the (a melanosome.multistage process The pH called of this maturation) organelle andvaries contributes during the to commondevelopment pigmentation of the organelle variation (a in multistagehuman skin, process hair, called and eye maturation) color. Biallelic and rare contributes variants to in common proteins criticalpigmentation for the productionvariation inof human melanin skin, (e.g., hair, TYR) and or eye in pHcolor. regulation Biallelic ofrare the variants melanosome in proteins (e.g., OCA2critical and for SLC45A2)the pro- ductionlead to of a significantmelanin (e.g., reduction TYR) inor melaninin pH regulation pigmentation of the in themelanosome skin, eyes, (e.g., and hairOCA2 and and give SLC45A2)rise to oculocutaneous lead to a significant albinism reduction (OCA) (OCA1,in melanin OCA2, pigmentation and OCA4, in respectively). the skin, eyes, Melanin and hairsynthesis and give is rise critical to oculocutaneous for the protection albinism of the (OCA) skin and (OCA1, eyes fromOCA2, ultraviolet and OCA4, radiation; respec- a tively).reduction Melanin in melanin synthesis synthesis is critical increases for the theprotection risk of skinof the cancers. skin and Furthermore, eyes from ultraviolet a dramatic radiation;reduction a reduction in melanin in productionmelanin synthesis in the increases eye is also the associated risk of skin with cancers. foveal Furthermore, hypoplasia, a reduceddramatic visual reduction acuity, in andmelanin photophobia production among in the individuals eye is also with associated OCA [10 with]. Taken foveal together, hy- poplasia,the link reduced between visual altered acuity, melanin and pigment photophobia production among and individuals disease is wellwith documented;OCA [10]. Takenhowever, together, it remains the link poorly between understood altered melani how then pigment pH of this production organelle and affects disease function is well of documented;proteins critical however, for the it maintenance remains poorly of organelleunderstood pH how [11]. the pH of this organelle